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Would anyone know how to go about obtaining samples of Salmonella or E. coli for purely educational purposes?
Note: I just want to look at the stuff under a microscope.
To be honest, you really shouldn't be buying such things if you're not prepared to handle them properly. If you work with a proper lab, you will be inindated with vendors trying to sell you these and many others.
But to answer your question, if you are looking to purchase many such items ATCC is an excellent resource. Not only will you have to meet regulatory requirements, but you will also have to deal with a great many MTA's depending on what you are trying to get.
In response to edit: Salmonella is actually not normally a serious pathogen to a healthy adult. It is, however, still a human pathogen and should be handled under BSL2 conditions. So if you have the funds and time to setup a BSL2 lab at your house, by all means go to town. And I hope you don't live with any children, the elderly, or otherwise immunocompromised individuals.
In response to further edits: If you just want to look at things under the scope, then I suggest you get started by making your own homemade culture plates. If you can get your hands on some Lysogeny Broth (LB), agar, and petri dishes then you can make lb plates like a researcher in a real lab (minus an autoclave but you could use a pressure cooker). These are items the public could get, but might be harder to come by.
I however will assume that you want to make your own plates from common household ingredients (I used to do this a lot as kid, whatever that says about me).
Making your own culture dishes:
1) The "plates" themselves.
- I recommend getting the smallest Tupperware like containers your can find that are microwave safe, dishwasher safe, and really make a good seal (water tight at the least). They really don't have to be big, and you will make your life easier if you buy a bunch to match.
- They need to be super clean. After running them through your dishwasher at home, put them all in a boiling pot of water until you are ready to pour in your "media." A mid-low simmer will work
Note that you won't probably be able to have perfect sterile technique at your house, but you should try to do what you can.
2) Your "Media" or Broth
I highly recommend using pectin as your gelling agent if you can't get your hands on some agar. After that comes plain unflavored gelatin, but I recommend pectin. You can get it at almost any grocery store and my recipe is made assuming that. You can determine how much you want to make, just scale up the ratios. I'm going to list things as rough volume by volume so you won't need a scale.
i) Make the powder first:
45mL of pectin;
4 bouillon cubes ground as finely as you can (you can get different types and results depending on if you start with vegetable, beef, or chicken. I recommend starting with vegetable.);
125mL of refined white sugar;
Additives (more on this later)
ii) Mix the powder into 500mL of water
- Before you mix in the powder, hear the water to 50-75oC (mid low heat), but not boiling. Mix the powder in while stirring until dissolved.
iii) Bring the mixture up to a boil.
Let it boil for about 2 min, stirring all the while.
iv) Cover the pot and let it cool for 4-5 min.
You by no means want it cool down to room temperature, you just want to get it a little below boiling. If you are at 55oC or bellow it's definitely time move on.
v) Dip/fill your "plates" 1/3 full of media using clean/sterile utensils and seal while hot but not boiling.
You may have noticed this seems an awful lot like canning, and that's because it is. Again you want to be as clean as possible in this step and of course try not to burn yourself on anything.
vi) Let your media plates set and chill over night in the refrigerator.
You really probably only have to wait a few hours if you are anxious to get going. You are going to want to store the plates that you are not using in the refrigerator anyway. Also if you made a bunch of plates, let the cool on a counter before you put them in the fridge so that you don't raise the temp of your fridge too much.
Remember the thing about additives? You can play around with adding different amounts and different types of salts, vitamins, food, or other items to see how they effect the cultures you get to grow. All kinds of fun there. First go round, leave them all out.
Now you have your own home made culture plates! The above recipe was empirically tested by my childhood, and repeated by my son's. Not all pectin from the store may be equal, this is with Ball Classic (I have no relation to the company). I'd love to know people's results in trying it, because I might need to re-think how/what I'm doing to get it to work.
Go around your house swabbing things with sterile swabs (you can get these at a drug store), and then streak them out on plate. While it will be a little different with a swab and homemade plate, the idea is similar to what is done here.
Leave your plate in a warm wet place, but not in direct sunlight. Ideal temp is going to be around 37, but you can leave them above and below that and still get things to grow. On top of our dryer is where I leave.
After a day or so you should have colonies you can streak out on a slide and look under a scope. You will find all kinds of things this way. If you want a more formal way to put things on a slide, you can check out dry and wet mounting from a Google search.
You are actually quite likely to run into E.coli if you sample enough "areas" this way. Obviously don't eat or huff the stuff that ends up growing. Maybe you can post some fun pictures of this if you try it.
How to Collect Samples and Test for Mold or Bacteria
Laboratory results are as good as the sample. The sample type taken generally depends on the purpose of the investigation. The importance of accurate results cannot be overstated. Test results change people’s lives.
Tip. Collecting a good sample for lab testing. There’s not much point using a lab analysis report to guide your microbial remediation decisions or recommendations unless the sample collected for analysis was truly representative of the total building contamination. The aim should always be to collect the most representative sample possible. See our course How To Take Mold Samples.
In this first section, you will learn how to safely collect and send samples for mold testing services.
- Cut 2-3 inches of clear scotch tape avoid touching the sticky side by holding the piece of tape by the edges
- Press the tape gently onto the surface you wish to test for mold growth
- Peel the tape off surface holding the tape by the edges only
- Apply sticky side of tape to the inside of the ziplock bag do not fold the tape
- Close bag and label the sample appropriately (put only one sample per bag)
- Fill the chain of custody form and send it together with the samples to us.
How To Collect Bulk Samples for Mold Testing:
- Wear suitable gloves
- Cut a small piece (about 4 square inches) of the suspect material (e.g., carpet, drywall, wallpaper, wood) taking care not to disturb the mold
- Place the sample inside a clean plastic bag (for example ziplock)
- Close the bag and label the sample appropriately
- Fill the chain of custody form and send it together with the samples to us.
How To Collect Swab Samples for Mold or Bacteria Testing:
Dry swabs are recommended for wet surfaces and wet swabs for dry surfaces.
- Wear suitable gloves
- Remove swab from tube (If using swabs with a wetting agent, drain most of it on the sides of the tube before sampling)
- Swab the test surface by rolling the swab lightly back and forth. For quantification of the amount of mold or bacteria on the test surface, swab a known surface area (for example, 100 square centimeters)
- After swabbing, insert the swab in the tube – Firmly close cap and label the sample appropriately
- Fill the chain of custody form and send it together with the samples to us.
How To Collect Air Samples (Non-Culture) for Airborne Mold Testing:
Various sampling cassettes can be used. Use the manufacturer’s instructions. Also ensure the pump has been calibrated to the appropriate flow rate for the type of cassette to be used.
Note: replace stickers on Air Sampling Cassettes once sampling is completed to prevent contamination.
How To Collect Air Samples For Culture Analysis:
Settle Plate Samples
- Select suitable agar media for sampling
- Place the plates at table-top level and remove the lids
- Leave the plates open for 0.5-4 hours
- Cover the plates and secure the lids with clear tape
- Label the plates with appropriate information
- Place the samples in a cooler/box ensuring the samples are not in contact with ice packs (to avoid having the samples frozen)
- Fill the chain of custody form and send it together with the samples to us for incubation and identification of the resulting mold or bacteria.
How To Collect Air Samples (culturable) for Mold or Bacteria Testing
Volumetric air samples for culture analyses are taken by impacting a known volume of air onto a suitable growth medium. Commonly used samples are Reuter Centrifugal Sampler (RCS) or the Anderson Single Stage Sampler. This process is particularly important if you request MBL’s bacteria testing service. Please refer to the manufacturer’s instructions on how to take samples using your sampler.
Key Points To Remember:
- Use a permanent marker to label the samples
- Complete a chain of custody form (sample submittal form) with the relevant information
- For air samples, record the flow rate and sampling time or the total air volume collected on the form
- Secure the samples and the chain of custody form in a shipping container
- For samples that do not require culturing, refrigeration is usually not needed when submitting the samples to the laboratory for analysis
- When collecting samples, write down and include with the sample(s) the following information, or download and fill out our Analysis Request Form:
- Company (if applicable)
- Mailing Address
- Telephone Number (voice) and fax number (if applicable)
- Email address (if any)
- Date sample taken
- The type of analysis required (if not sure call the lab)
- Turnaround time required for non-viable analysis: regular (1-3 days) or rush (24 hours
If you are within the GTA region, you can either deliver the samples to the laboratory by hand or send them by courier or post. Samples from anywhere else in Canada (outside the GTA) may be sent by courier or post.
Note: It is recommended that wet and/or culturable samples be sent to the laboratory on the same day if possible) or by overnight courier, and should be shipped under cool conditions (but not frozen).
When we test for moulds or bacteria, our turnaround time for all culture analyses is 10-14 days. Non-culture analyses takes 1-3 days for regular service and 24 hours for rush service.
A. STARCH HYDROLYSIS
Starch is a polysaccharide which appears as a branched polymer of the simple sugar glucose. This means that starch is really a series of glucose molecules hooked together to form a long chain. Additional glucose molecules then branch off of this chain as shown below.
( ---GLU-GLU-GLU-GLU-GLU-GLU-GLU--- )n
Some bacteria are capable of using starch as a source of carbohydrate but in order to do this, they must first hydrolyze or break down the starch so it may enter the cell. The bacterium secretes an exoenzyme which hydrolyzes the starch by breaking the bonds between the glucose molecules. This enzyme is called a diastase.
( ---GLU / GLU / GLU / GLU / GLU / GLU / GLU--- )n
action of diastase
The glucose can then enter the bacterium and be used for metabolism.
Trypticase Soy broth cultures of Bacillus subtilis and Escherichia coli.
PROCEDURE (to be done in pairs)
1. Using a wax marker, draw a line on the bottom of a Starch agar plate so as to divide the plate in half. Label one half B. subtilis and the other half E. coli.
2. Make a single streak line with the appropriate organism on the corresponding half of the plate as shown in Fig. 6.
3. Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section until the next lab period.
4. Next period, iodine will be added to see if the starch remains in the agar or has been hydrolyzed by the exoenzyme diastase. Iodine reacts with starch to produce a dark brown or blue/black color. If starch has been hydrolyzed there will be a clear zone around the bacterial growth (see Fig. 3A) because the starch is no longer in the agar to react with the iodine. If starch has not been hydrolyzed, the agar will remain a dark brown or blue/black color (see Fig. 3B).
B. PROTEIN HYDROLYSIS
Proteins are made up of various amino acids linked together in long chains by means of peptide bonds. Many bacteria can hydrolyze a variety of proteins into peptides (short chains of amino acids) and eventually into individual amino acids. They can then use these amino acids to synthesize their own proteins and other cellular molecules or to obtain energy. The hydrolysis of protein is termed proteolysis and the enzyme involved is called a protease. In this exercise we will test for bacterial hydrolysis of the protein casein, the protein that gives milk its white, opaque appearance.
Trypticase Soy broth cultures of Bacillus subtilis and Escherichia coli.
PROCEDURE (to be done in pairs)
1. Divide the Skim Milk agar plate in half and inoculate one half with Bacillus subtilis and the other half with Escherichia coli as done above with the above starch agar plate (see Fig. 7).
2. Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section until the next lab period. If casein is hydrolyzed, there will be a clear zone around the bacterial growth (see Fig 1A). If casein is not hydrolyzed, the agar will remain white and opaque (see Fig. 1B).
C. FERMENTATION OF CARBOHYDRATES
Carbohydrates are complex chemical substrates which serve as energy sources when broken down by bacteria and other cells. They are composed of carbon, hydrogen, and oxygen (with hydrogen and oxygen being in the same ratio as water [CH 2 O]) and are usually classed as either sugars or starches.
Facultative anaerobic and anaerobic bacteria are capable of fermentation, an anaerobic process during which carbohydrates are broken down for energy production. A wide variety of carbohydrates may be fermented by various bacteria in order to obtain energy and the types of carbohydrates which are fermented by a specific organism can serve as a diagnostic tool for the identification of that organism.
We can detect whether a specific carbohydrate is fermented by looking for common end products of fermentation. When carbohydrates are fermented as a result of bacterial enzymes, the following fermentation end products may be produced:
1. acid end products, or
2. acid and gas end products .
In order to test for these fermentation products, you inoculate and incubate tubes of media containing a single carbohydrate (such as lactose or maltose), a pH indicator (such as phenol red) and a Durham tube (a small inverted tube to detect gas production).
- If the bacterium ferments that particular carbohydrate producing acid end products alone, the acid will lower the pH, causing the pH indicator phenol red to change form its original red color at a neutral pH to a yellow or clear colorsee Fig. 2A).
- If the bacterium ferments that particular carbohydrate producing both acid and gas, the pH indicator phenol red to change form its original red color at a neutral pH to a yellow or clear color and the gas will collect in the Durham tube as a substantial gas bubble appearing at the top of the Durham tube(see Fig. 2B) .
- If the carbohydrate is not fermented by the bacterium, no acid or gas will be produced and the phenol red will remain red(see Fig. 2C).
3 tubes of Phenol Red Lactose broth and 3 tubes of Phenol Red Maltose broth (see Fig. 4D)
Trypticase Soy agar cultures of Bacillus subtilis, Escherichia coli, and Staphylococcus aureus.
PROCEDURE (to be done in pairs)
1. Label each tube with the name of the sugar in the tube and the name of the bacterium you are growing.
2. Inoculate one Phenol Red Lactose broth tube and one Phenol Red Maltose broth tube with Bacillus subtilis.
3. Inoculate a second Phenol Red Lactose broth tube and a second Phenol Red Maltose broth tube with Escherichia coli.
4. Inoculate a third Phenol Red Lactose broth tube and a third Phenol Red Maltose broth tube with Staphylococcus aureus.
5 . Incubate the tubes in your test tube rack on your shelf of the 37°C incubator corresponding to your lab section until the next lab period
D. INDOLE AND HYDROGEN SULFIDE PRODUCTION
Sometimes we look for the production of products produced by only a few bacteria. As an example, some bacteria use the enzyme tryptophanase to convert the amino acid tryptophan into molecules of indole, pyruvic acid and ammonia. Since only a few bacteria contain tryptophanase, the formation of indole from a tryptophan substrate can be another useful diagnostic tool for the identification of an organism. Indole production is a key test for the identification of Escherichia coli.
By adding Kovac's reagent to the medium after incubation we can determine if indole was produced. Kovac's reagent will react with the indole and turn red (see Fig. 5A).
Likewise, some bacteria are capable of breaking down sulfur containing amino acids (cystine, methionine) or reducing inorganic sulfur-containing compounds (such as sulfite, sulfate, or thiosulfate) to produce hydrogen sulfide (H 2 S). This reduced sulfur may then be incorporated into other cellular amino acids, or perhaps into coenzymes. The ability of an organism to reduce sulfur-containing compounds to hydrogen sulfide can be another test for identifying unknown organisms such as certain Proteus and Salmonella. To test for hydrogen sulfide production, a medium with a sulfur-containing compound and iron salts is inoculated and incubated. If the sulfur is reduced and hydrogen sulfide is produced, it will combine with the iron salt to form a visible black ferric sulfide (FeS) in the tube (see Fig. 5B) .
If neither hydrogen sulfide nor indole are produced in SIM medium, the agar does not turn black and the Kovac's reagent remains yellow. (Fig. 5C).
Three tubes of SIM (Sulfide, Indole, Motility) medium (see Fig. 5C). This medium contains a sulfur source, an iron salt, the amino acid tryptophan, and is semi-solid in agar content (0.3%). It can be used to detect hydrogen sulfide production, indole production, and motility.
Trypticase Soy agar cultures of Proteus mirabilis, Escherichia coli, and Enterobacter cloacae.
PROCEDURE (to be done in pairs)
1. Stab one SIM medium tube with Proteus mirabilis.
2. Stab a second SIM medium tube with Escherichia coli.
3. Stab a third SIM medium tube with Enterobacter cloacae.
4 . Incubate the tubes in your test tube rack on your shelf of the 37°C incubator corresponding to your lab section until the next lab period
5. Next lab period add Kovac's reagent to each tube to detect indole production.
E. CATALASE ACTIVITY (Demonstration)
Catalase is the name of an enzyme found in most bacteria which initiates the breakdown of hydrogen peroxide (H 2 O 2 ) into water (H 2 O) and free oxygen (O 2 ).
During the normal process of aerobic respiration, hydrogen ions (H + )are given off and must be removed by the cell. The electron transport chain takes these hydrogen ions and combines them with half a molecule of oxygen (an oxygen atom) to form water (H 2 O). During the process, energy is given off and is trapped and stored in ATP. Water is then a harmless end product. Some cytochromes in the electron transport system, however, form toxic hydrogen peroxide (H 2 O 2 ) instead of water and this must be removed. This is done by the enzyme catalase breaking the hydrogen peroxide into water and oxygen as shown above. Most bacteria are catalase-positive however, certain genera that don't carry out aerobic respiration, such as the genera Streptococcus, Enterococcus, Lactobacillus, and Clostridium, are catalase-negative.
Trypticase Soy agar cultures of Staphylococcus aureus and Streptococcus lactis, 3% hydrogen peroxide.
Add a few drops of 3% hydrogen peroxide to each culture and look for the release of oxygen as a result of hydrogen peroxide breakdown. This appears as foaming. (see Fig. 5A)
A. Starch Hydrolysis
When iodine is added to starch, the iodine-starch complex that forms gives a characteristic dark brown or deep purple color reaction. If the starch has been hydrolyzed into glucose molecules by the diastase exoenzyme, it no longer gives this reaction.
Flood the surface of the Starch agar plate with gram's iodine.
- If the bacterium produced an exoenzyme that hydrolized the starch in the agar, aclear zone will surrround the bacterial growth because the starch is no longer there to react with the iodine(see Fig. 3A).
- If the bacterium lacks the exoenzyme to break down the starch, the agar around the growth should turn dark brown or blue/black color (see Fig. 3B)due to the iodine-starch complex.
Record your results and indicate which organism was capable of hydrolyzing the starch (+ = hydrolysis - = no hydrolysis).
Escherichia coli Bacillus subtilis Starch hydrolysis = Starch hydrolysis =
B. Protein Hydrolysis
The protein casein exists as a colloidal suspension in milk and gives milk its characteristic white, opaque appearance. If the casein in the milk is hydrolyzed into peptides and amino acids, it will lose its opaqueness.
- If the bacterium produced an exoenzyme capable of hydrolyzing the casein, there will be aclear zone around the bacterial growth(see Fig. 1A).
- If the bacterium lacks the exoenzyme to break down casein, the Skim Milk agar will remainwhite and opaque(see Fig. 1B).
Record your results and indicate which organism was capable of hydrolyzing casein (+ = hydrolysis - = no hydrolysis).
Escherichia coli Bacillus subtilis Casein hydrolysis = Casein hydrolysis =
C. Fermentation of Carbohydrates
As mentioned above, w e can detect whether a specific carbohydrate is fermented by looking for common end products of fermentation. When carbohydrates are fermented as a result of bacterial enzymes, the following fermentation end products may be produced:
1. acid end products, or
2. acid and gas end products .
The results of fermentation may be acid alone or acid plus gas, but never gas alone.
Phenol red pH indicator appears red or orange at neutral pH and appears yellow or clear at an acidic pH.
- A change in color in the tube from red or orange to yellow or clear indicates that the organism has fermented that particular carbohydrate, producing acid end products.
- A substantial gas bubble at the top of the Durham tube, the inverted test tube within the broth, indicates gas was also produced from the fermentation of the carbohydrate.
- If the phenol red remains red, no acid was produced and the carbohydrate was not fermented.
Possible results are as follows:
- Carbohydrate fermentation producing acid but no gas: acidic (yellow or clear) no substantial gas bubble in the Durham tube (see Fig. 2A) .
- Carbohydrate fermentation producing acid and gas: acidic (yellow or clear) a substantial gas bubble in the Durham tube (see Fig. 2B) .
- No carbohydrate fermentation. No acid or gas (neutral pH (red or orange) no substantial gas bubble in the Durham tube (see Fig. 2C) .
D. Production of Indole and Hydrogen Sulfide
Carefully add about 1/4 inch of Kovac's reagent to each of the 3 SIM agar tubes and observe.
1. Production of hydrogen sulfide (H 2 S)
- If the bacterium produces the enzyme to reduce sulfur to hydrogen sulfide (H 2 S), the agar will turn black indicating that the organism has produced hydrogen sulfide(see Fig. 5B).
- If the bacterium lacks the enzyme, the agar does not turn black, indicating that hydrogen sulfide was not produced.
2. Production of indole
- If the bacterium produces the enzyme to break down tryptophan into molecules of indole, pyruvic acid, and ammonia, the Kovac's reagent will turn red, indicating the organism is indole-positive(see Fig. 5A).
- If the Kovac's reagent remains yellow, no indole was produced and the organism is indole-negative(see Fig. 5C).
E. Catalase Activity
Catalase is the name of an enzyme found in most bacteria which initiates the breakdown of hydrogen peroxide (H2O2) into water and free oxygen.
- If the bacterium produces the enzyme catalase, then the hydrogen peroxide added to the culture will be broken down into water and free oxygen. The oxygen will bubble through the water causing a surface froth to form. This is a catalase-positive bacterium (see fig. 6A).
- A catalase-negativebacterium will not produce catalase to break down the hydrogen peroxide, and no frothing will occur (see Fig. 6B).
PERFORMANCE OBJECTIVES FOR LAB 8
After the completion of this lab, the student will be able to complete the following objectives:
1. State the chemical nature and function of enzymes.
2. Define endoenzyme and exoenzyme.
A. STARCH HYDROLYSIS
1. Describe a method of testing for starch hydrolysis and state how to interpret the results.
1. Interpret the results of starch hydrolysis on a Starch agar plate that has been inoculated, incubated, and flooded with iodine.
B. PROTEIN HYDROLYSIS
1. Describe a method of testing for casein hydrolysis and state how to interpret the results.
1. Interpret the results of casein hydrolysis on a Skim Milk agar plate after it has been inoculated and incubated.
C. FERMENTATION OF CARBOHYDRATES
1. Name the general end products which may be formed as a result of the bacterial fermentation of sugars and describe how these end products change the appearance of a broth tube containing a sugar, the pH indicator phenol red, and a Durham tube.
1. Interpret the carbohydrate fermentation results in tubes of Phenol Red Carbohydrate broth containing a Durham tube after it has been inoculated and incubated.
D. INDOLE AND HYDROGEN SULFIDE PRODUCTION
1. State the pathway for the breakdown of tryptophan to indole. 2. State the pathway for the detection of sulfur reduction in SIM medium.
3. State three reactions that may be tested for in SIM medium and describe how to interpret the results.
1. Interpret the hydrogen sulfide and indole results in a SIM medium tube after inoculation, incubation, and addition of Kovac's reagent.
E. CATALASE ACTIVITY
1. State the function of the enzyme catalase and describe a method of testing for catalase activity.
1. Interpret the results of a catalase test after adding hydrogen peroxide to a plate culture of bacteria.
How To Grow Bacteria and More
If learning how to grow bacteria interests you, read on.
Bacteria are one-celled, or unicellular, microorganisms. They are different from plant and animal cells because they don’t have a distinct, membrane-enclosed nucleus containing genetic material. Instead, their DNA floats in a tangle inside the cell. Are viruses protists?
Individual bacteria can only be seen with a microscope, but they reproduce so rapidly that they often form colonies that we can see. Bacteria reproduce when one cell splits into two cells through a process called binary fission. Fission occurs rapidly in as little as 20 minutes. Under perfect conditions, a single bacterium could grow into over one billion bacteria in only 10 hours! (It’s a good thing natural conditions are rarely perfect, or the earth would be buried in bacteria!)
Growing and testing bacteria is a fun any-time project or a great science fair project. Bacteria are everywhere, and since they reproduce rapidly they are easy to study with just a few simple materials. All you need are some petri dishes, agar, and sterile swabs or an inoculating needle. Agar is a gelatinous medium that provides nutrients and a stable, controlled environment for bacteria growth (agar solution). Most bacteria will grow well-using nutrient agar, but some more fastidious bacteria (those with more complex nutrient requirements like Bacillus stearothermophilus, Branhamella catarrhalis, and Bacillus coagulans) prefer tryptic soy agar.
You also need a source for bacteria, and this is not hard to find! You can swab your mouth or skin, pets, soil, or household surfaces like the kitchen sink or toilet bowl. If you want to study a particular type of bacteria, you can also purchase live cultures in our microbiology for kids section. Keep reading to see four experiments using bacteria and more ideas for science projects (also consider this hands-on bacteria growing kit)! Adult supervision is recommended when working with bacteria.
How Can Bacteria Help Us?
Where would we be without bacteria? Well, we might not be getting bacterial diseases, but we would still be a lot worse off! Bacteria perform all sorts of very important functions, both in our bodies and in the world around us. Here are just a few.
Digestion. Our large intestines are full of beneficial bacteria that break down food that our bodies can’t digest on their own. Once the bacteria break it down, our intestines are able to absorb it, giving us more nutrients from our food.
Vitamins. Bacteria in our intestines actually produce and secrete vitamins that are important for our health! For example, E. coli bacteria in our intestines are a major source of vitamin K. (Most E. coli is good for us, but there is a harmful type that causes food poisoning.)
Food. Bacteria are used to turn milk into yogurt, cheese, and other dairy products.
Oxygen. Cyanobacteria (which used to be called blue-green algae) live in water and perform photosynthesis, which results in the production of much of the oxygen we need to breathe.
Cleanup. Oil spills, sewage, industrial waste — bacteria can help us clean all of these up! They ‘eat’ the oil or toxins and convert them into less harmful substances.
Bacteria are amazing creatures, aren’t they? They can be so dangerous and yet so important at the same time. Keep reading to see an experiment that uses good bacteria!
How Can Bacteria Harm Us?
Some types of bacteria cause disease and sickness. These kinds of bacteria are called pathogens. They reproduce very rapidly, like all bacteria. These come in many forms and can cause illnesses from an ear infection to strep throat to cholera. They can get into our bodies via our mouth and nose or through cuts and scrapes. Some are airborne, others are found in food, resulting in food poisoning. Bacteria are also the cause of plaque buildup on our teeth, which can lead to cavities and gum disease.
Before the discovery of antibiotics, many severe bacterial diseases had no cure and usually resulted in death. Antibiotics work by destroying bacteria or inhibiting their reproduction while leaving the body’s own cells unharmed. After a time, some bacteria develop resistance to an antibiotic, and it will no longer be effective against them. Because of this, scientists are always researching new antibiotics. (Many diseases, such as chickenpox, hepatitis, or polio, are caused by viruses rather than bacteria. Antibiotics have no effect against these diseases.)
Bacterial infections are common, but many of them can be avoided by good cooking, cleaning, and hand-washing practices.
What Are Antibacterial Agents?
How do people stop bacteria from growing and spreading? They control it in two ways: by killing the bacteria cells, and by stopping the bacteria from reproducing. An agent is a solution or method which either kills or stops reproduction. Bactericides are agents that kill bacteria cells. Static agents inhibit cell growth and reproduction.
There are a variety of ways to kill bacteria or keep it from reproducing.
- Sterilization. The application of heat to kill bacteria. Includes incineration (burning), boiling, and cooking.
- Pasteurization. The use of mild heat to reduce the number of bacteria in food.
- Cold temperatures. Refrigeration and freezing are two of the most common methods used in homes, for preserving food’s life span.
- Antiseptics. These agents can be applied directly to living tissues, including human skin.
- Disinfectants. These agents are not safe for live tissues. Disinfectants are used to clean toilets, sinks, floors, etc.
- Preservatives. These are used in almost every processed food that is available today. They inhibit bacterial growth in foods.
- Some food preservatives are sodium benzoate, monosodium glutamate (MSG), sulfur dioxide, salts, sugar, and wood smoke.
- Amoxycillin and Ampicillin—inhibit steps in cell wall synthesis (building)
- Penicillin—inhibits steps in cell wall synthesis
- Erythromycin—inhibits RNA translation for protein synthesis
While most environmental bacteria are not harmful to healthy individuals, once concentrated in colonies, they can be hazardous.
To minimize risk, wear disposable gloves while handling bacteria, and thoroughly wash your hands before and after. Never eat or drink during bacteria studies, nor inhale or ingest growing cultures. Work in a draft-free room and reduce airflow as much as possible. Keep petri dishes with cultured mediums closed—preferably taped shut—unless sampling or disinfecting. Even then, remove the petri dish only enough to insert your implement or cover medium with bleach or 70% isopropyl alcohol.
When finished experimenting, seal dishes in a plastic bag and dispose. Cover accidental breaks or spills with bleach or alcohol for 10 minutes, then carefully sweep up, seal in a plastic bag, and discard.
Preparing Culture Dishes
Before you can grow bacteria, you’ll need to prepare sterile culture dishes. A 125ml bottle of nutrient agar contains enough to fill about 10 petri dishes.
Water Bath Method – Loosen the agar bottle cap, but do not remove it completely. Place the bottle in hot water at 170-190 °F until all of the agar is liquid. To prevent the bottle from tipping, keep the water level even with the agar level.
- Let the agar cool to 110-120 °F (when the bottle still feels warm but not too hot to touch) before pouring into petri dishes.
- Slide open the cover of the petri dish just enough to pour agar into the dish. Pour enough agar to cover 1/2 to 2/3 of the bottom of the dish (about 10-13ml). Don’t let the bottle mouth touch the dish. Cover the dish immediately to prevent contamination and tilt it back and forth gently until the agar coats the entire bottom of the dish. (Fill as many dishes as you have agar for: you can store extras upside down until you’re ready to use them.)
- Let the petri dishes stand one hour for the agar to solidify before using them.
Experiment #1: Cheek Cell Swab
Make a culture dish using the instructions above. Once the culture dish is prepared, use a sterile cotton swab or inoculating needle and swab the inside of your cheek. Very gently rub the swab over the agar in a few zigzag strokes and replace the lid on the dish. You’ll need to let the dish sit in a warm area for 3-7 days before bacteria growth appears. Record the growth each day with a drawing and a written description. The individual bacteria are too tiny to see without a high-power microscope, but you can see bacteria colonies. Distinguish between different types of bacteria by the color and shape of the colonies.
Experiment #2: Testing Antibacterial Agents
Preparing Sensitivity Squares
One method for testing the antibacterial effectiveness of a substance is to use ‘sensitivity squares.’ Cut small squares of blotter paper (or other absorbent paper) and then soak them in whatever substance you want to test: iodine, ethyl alcohol, antibacterial soap, antiseptics, garlic, etc. Use clean tweezers to handle the squares so you don’t contaminate them. Label them with permanent ink, soak them in the chosen substance, and blot the excess liquid with a paper towel.
Decide on a source for collecting bacteria. For using sensitivity squares, make sure there is just one source, and keep each dish as consistent as possible. Sources could include a kitchen sink, bathroom counter, cell phone, or another surface you would like to test. Rub a sterile swab across the chosen surface, and then lightly rub it across the prepared agar dish in a zigzag pattern. Rotate the dish and repeat.
Setting Up an Experiment
Each experiment should have a control dish that shows bacteria growth under normal conditions and one or more test dishes in which you change certain variables and examine the results. Examples of variables to test are temperature or the presence of antiseptics. How do these affect bacteria growth?
- Label one dish ‘Control.’ Then in your test dish, use tweezers to add the sensitivity squares that have been soaked in a substance you wish to test for antibacterial properties. It’s a good idea to add a plain square of blotter paper to see if the paper by itself has any effect on bacteria growth. For best results, use multiple test dishes and control the variables so the conditions are identical for each dish: bacteria collected from the same place, exposed to the same amount of antibacterial substance, stored at the same temperature, etc. The more tests you perform, the more data you will collect, and the more confident you can be about your conclusions.
- Place all the dishes in a dark, room-temperature place like a closet.
Wait 3-7 days and examine the bacteria growth in the dishes, without removing the lids. You will see multiple round dots of growth these are bacteria colonies. Depending on where you collected your bacteria samples, you may have several types of bacteria (and even some mold!) growing in your dishes. Different types of colonies will have different colors and textures. If you have a compound or stereo microscope, try looking at the colonies up close to see more of the differences.
Compare the number of bacteria in the control dish to the amount in the test dishes. Next, compare the amount of bacteria growth around each paper square. Which one has bacteria growing closest to it? Which one has the least amount of bacteria growing near it? If you did more than one test dish, are the results similar in all the test dishes? If not, what variables do you think might have caused the results to be different? How does this affect your conclusions?
For a variation on this experiment, test the effect of temperature on bacterial growth instead of using sensitivity squares. Put a control dish at room temperature, and place other dishes in dark areas with different temperatures.
Experiment #3: Soap Survey
Every time you touch something you are probably picking up new bacteria and leaving some behind. This is how many infectious diseases spread — we share our bacteria with everyone around us! Even bacteria that lives safely on our skin can make us sick if it gets inside our bodies through our mouths or cuts and scrapes. This is one reason why it is so important that we wash our hands frequently and well.
What kind of soap works best for cutting down on the bacteria on our hands? You can test this by growing some bacteria cultures using agar and petri dishes.
- Two (or more) petri dishes or other absorbent paper or tweezers
- Different kinds of hand cleaners: regular soap, antibacterial soap, dish soap, hand sanitizer
- Prepare the agar according to the directions on the label, then pour enough to cover the bottom of each petri dish. Cover the dishes and let them stand for about an hour until the agar has solidified again. (If you aren’t going to use them right away after they have cooled, store them upside down in the refrigerator.)
- When your petri dishes are ready, collect some bacteria from your hand or the hand of a volunteer. (Make sure the person hasn’t washed his or her hands too recently!) Do this by rubbing the sterile swab over the palm in a zigzag pattern.
- Remove the cover from the petri dish and lightly rub the swab back and forth in a zigzag pattern on the agar. Turn the dish a quarter turn and zigzag again. Cover the dish and repeat steps two and three for the other dish, using a new sterile swab. Label the dishes “Test” and “Control.” (You may want to do more than one test dish, so you can compare the results.)
- Cut the blotter paper into small “sensitivity squares.” Use permanent ink to label the squares for the different types of hand cleaners you are going to test, e.g., “R” for regular soap, “A” for antibacterial soap, and “S” for hand sanitizer. Using tweezers, dip each square into the appropriate cleaner. Blot the excess cleaner on a paper towel and then place the squares on the agar in the “Test” dish. (Spread the squares out so there is distance between them.) Add one square of plain blotter paper to test if blotter paper by itself has any effect. Don’t put any squares in the “Control” dish – this one will show you what the bacterial growth will look like without any soap.
- Put the dishes in a dark, room-temperature place like a closet and leave them undisturbed for a few days.
The rate of bacteria growth in your dishes will depend on temperature and other factors. Check your cultures after a couple of days, but you’ll probably want to wait 5-7 days before recording your data. You will see multiple round dots of growth these are bacteria colonies. There may be several types of bacteria growing in the dishes. Different types of colonies will have different colors and textures.
For each soap test, count and record the number of bacteria colonies in each dish. To see how effective each soap was, divide the number of colonies in the test dish by the number of colonies in the control dish, then subtract the result from 1 and write the answer as a percentage. For example, if your control dish had 100 colonies and your soap test dish had 30, the soap eliminated 70% of the bacteria: 1 — (30 ÷ 100) = .7 = 70%
According to your results, which type of soap was the most effective at eliminating bacteria? Does “antibacterial” soap really work better than regular soap? How well did washing hands in water without soap work? What further tests could you do to determine which soaps and hand washing methods are most effective at eliminating bacteria?
Experiment #4: Bacteria in the Air
You need two culture dishes for this experiment, in which you’ll demonstrate how antibacterial agents (such as antibiotics and household cleaners) affect bacteria growth.
Leave the dishes with their lids off in a room-temperature location. Leave the culture dishes exposed for about an hour.
While you wait, cut small squares of paper (blotter paper works well), label them with the names of the antibacterials you’re going to test (e.g. ‘L’ for Lysol, ‘A’ for alcohol, etc.), and soak each in a different household chemical that you wish to test for antibacterial properties. If you have time, you might also experiment with natural antibacterial agents, such as tea tree oil or red pepper. Wipe off any excess liquid and use tweezers to set each of the squares on a different spot in one of the culture dishes. The second culture dish is your ‘control.’ It will show you what an air bacteria culture looks like without any chemical agents.
Store the dishes (with lids on) in a dark place like a closet where they will be undisturbed for a few days. After 3-7 days, take both culture dishes and carefully observe the bacteria growth in each dish, leaving the lids on. The bacteria will be visible in small, colored clusters. Take notes of your observations and make drawings. You could also answer the following questions. In the control culture, How much of the dish is covered with bacteria? In the sensitivity square test culture, Have the bacteria covered this dish to the same extent as the control culture? What effect have each of the chemicals had on the bacteria growth? Did a particular chemical kill the bacteria or just inhibit its growth?
- For further study, you could use an antibiotic disc set to see what different antibiotics can do against bacteria.
- For a more advanced project, learn how gram staining relates to the use of antibiotics.
Experiment #5: Homemade Yogurt
Generally when people think of ‘bacteria,’ they think of harmful germs. However, not all forms of bacteria are bad!
You can enjoy a tasty product of good bacteria by making a batch of yogurt at home.
You’ll need to use a starter (available at grocery or health food stores), or else one cup of plain, unflavored yogurt that has live cultures in it. (If it contains live cultures, it will say so on the container.)
Slowly heat four cups of milk until it is hot, but not boiling or scalding. The temperature should be around 95-120 degrees to kill some of the harmful bacteria. Cool slightly, until milk is warm, and then add one cup of active yogurt or the starter.
Put the mixture in a large bowl (or glass jars) and cover. Make sure that the bowl or jars are sterilized before using by either running them through the dishwasher or washing them with very hot water.
There are two different methods for culturing the yogurt mixture: You can put the covered bowl or jars into a clean plastic cooler, and fill the cooler with hot water to just below the top of the culture containers. With this method, you will need to occasionally refill the cooler with hot water, so that the temperature of the yogurt stays consistent. The other method is to wrap the containers in a heating pad and towels, setting the heating pad on low to medium heat.
Check the mixture after heating for 3 1/2 to 4 hours. It should be ‘set up,’ having a smooth, creamy consistency similar to store-bought yogurt. If the mixture is not set up yet, heat it for another 1-2 hours. When it is the right consistency, add some flavoring—such as vanilla extract, chocolate syrup, or berries—and store the yogurt in the refrigerator. It should keep for a couple of weeks. For safety, we suggest that you do not eat any yogurt that has separated or has a non-typical consistency.
Energy Sources for Bacteria
As bacteria are living organisms, it is clear that they get the substances required for the production of energy and for cellular biosynthesis from the environment in which they thrive. The essential substances pass into and out of the bacterial cell membranes. Bacteria obtain food from the environment, and can break the food down. Various bacteria obtain food in various ways.
These are the organisms that synthesize their own organic food. These bacteria use inorganic substances to produce their organic food. They get carbon from carbon dioxide and they use hydrogen obtained from hydrogen sulfide (H2S) or ammonia (NH3) or hydrogen (H2). Autotrophic bacteria are further divided into phototrophs and chemotrophs (lithotrophs, organotrophs).
Phototrophs: These bacteria have photosynthetic pigments called ‘bacteriochlorophyll’ (like chlorophyll in plants) in the membranes. They harness the sun’s light to make food and generate energy. They do not produce oxygen during photosynthesis (plants do). Cyanobacteria, Green sulfur bacteria, Chloroflexi or Purple bacteria are examples of photrophs.
Lithotrophs: Inorganic compounds are the main source of energy for lithotrophs. These bacteria get their nutrients (inorganic compounds) from the minerals in rocks. Bacteria are formed of carbon, oxygen, nitrogen, hydrogen and phosphorus. They also consist of traces of other elements. So they need to obtain these nutrients from the environment for survival. Lithotrophs get most of these nutrients from rocks.
Inorganic compounds like hydrogen sulfide, elemental sulfur, ammonium and ferrous iron are oxidized by lithotrophs in order to obtain energy. Nitrifying bacteria (Nitrosomonas, Nitrobacter) derive energy by oxidizing ammonia into nitrates. Sulfur bacteria (Thiobacillus, Beggiatoa) gain energy by oxidizing hydrogen sulfide to sulfur. Oxidization of ferrous ions into ferric form gives energy to iron bacteria (Ferrobacillus, Gallionella). But lithotrophs do not get carbon from the minerals in the rocks. Some lithotrophs get carbon from the air, while some get it from the organic matter.
Organotrophs: These bacteria get their nutrients and generate energy from the organic compounds. For survival, they consume autotrophic or heterotrophic organisms, milk, meat, and decaying materials (remains). Pathogenic bacteria belong to this group. They live in the body of animals and plants, and get their organic food from there. Bacillus, Clostridium or Enterobacteriaceae are examples of organotrophs.
These bacteria consume food that is already present in the environment. This means that they are not able to synthesize their own organic food. In autotrophic bacteria, cellular carbon is obtained by fixing carbon dioxide. In heterotrophic bacteria, organic carbon compounds provide carbon to the bacteria. These include the parasitic types of bacteria.
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Saprophytic Bacteria: These are the bacteria that obtain nutrients from dead organic matter. The exogenous enzymes secreted by these bacteria promote the breakdown of the complex organic matter into easily absorbable (soluble) form. Thus, they absorb the nutrients which help generate energy. These bacteria are considered as friendly bacteria, as they play an important role in the ecosystem by working as decomposers.
Aerobic and Anaerobic Bacteria: Bacteria can decompose organic material, and this property is used in the food industry for ripening of cheese, in loosening of fiber, in curing of tobacco, etc. The aerobic breakdown of organic matter is known as decay or decomposition. The anaerobic breakdown of organic matter is termed as fermentation. You can read this article about aerobic vs anaerobic bacteria, for more information.
Researchers had to work a lot to find an answer to the question as to how do bacteria obtain energy. The study of bacteria is known as bacteriology. If you are interested in studying bacteria, you can choose microbiology for graduation, and then for specialization, you can choose bacteriology.
Streptomyces, lactobacillis and E. coli are some examples of helpful bacteria. To know more about the species of beneficial bacteria, read on.
Aerobic bacteria require oxygen to perform cellular respiration and derive energy to survive. In short, aerobic bacteria grows and multiplies only in the presence of oxygen. To know more about&hellip
In this BiologyWise article, we put forth the differences between aerobic and anaerobic bacteria in order to make it easier for you to understand their characteristics.
Our editors will review what you’ve submitted and determine whether to revise the article.
Sulfur bacterium, plural Sulfur Bacteria, any of a diverse group of microorganisms capable of metabolizing sulfur and its compounds and important in the sulfur cycle (q.v.) in nature. Some of the common sulfur substances that are used by these bacteria as an energy source are hydrogen sulfide (H2S), sulfur, and thiosulfate (S2O3 2- ). The final product of sulfur oxidation is sulfate (SO4 2- ).
Thiobacillus, widespread in marine and terrestrial habitats, oxidizes sulfur, producing sulfates useful to plants in deep ground deposits it generates sulfuric acid, which dissolves metals in mines but also corrodes concrete and steel. Desulfovibrio desulficans reduces sulfates in waterlogged soils and sewage to hydrogen sulfide, a gas with the rotten egg odour so common to such places. Thiothrix, common in sulfur springs and in sewage, and Sulfolobus, confined to sulfur-rich hot springs, transform hydrogen sulfide to elemental sulfur.
Many species in the families Chromatiaceae (purple sulfur bacteria) and Chlorobiaceae (green sulfur bacteria) utilize energy from light in an oxygen-free environment to transform sulfur and its compounds to sulfates.
Transport Media Used in Microbiology Lab
Transport media are essentially solutions of buffers with carbohydrates, peptones and other nutrients (excluding growth factors) designed to preserve the viability of bacteria during transport without allowing their multiplication. The primary objective of the use of the transport medium is to maintain the specimen as near its original state as possible.
Transport medium aims to preserve a specimen and minimize bacterial overgrowth from the time of collection to the processing of the specimen. Depending on the type of organisms suspected in the sample, transport media may vary.
However, in general, transport media are classified on the basis of the physical state as semi-solid and liquid and also on the basis of their utility as bacterial or viral transport media.
- Contains only buffers and salt.
- Doesn’t contain any nutritional ingredients such as carbon, nitrogen, and organic growth factors so as to prevent microbial multiplication.
- Addition of antibiotics and other substances like glycerol may be added for transporting specimens for tissue culture.
What samples are collected in transport medium?
All types of samples that may contain pathogens but could not be processed immediately require transport media. It may be stool, urethral swabs, nasal and throat swabs and specimens for tissue culture, etc.
What are the commonly used Transport media?
- Cary and Blair Medium: semi-solid, white-colored transport medium for feces that may contain Salmonella, Shigella, Vibrio or CampylobacterCharcoal helps eliminate metabolic products of bacterial growth, which may be especially useful in the isolation of fastidious organisms like However it is suggested that, some other pathogens like Campylobacter can also survive in such medium.
- Amies medium without charcoal: Are ideal for the isolation of Mycoplasma and Ureaplasma
- Stuarts medium: Commonly used for transporting specimens suspected of having gonococci. Also used for transporting throat, wound and skin swabs that may contain fastidious organisms.
- Venkatraman Ramakrishnan (VR) medium: Used to transport feces from suspected cholera patients.
- Sach’s buffered glycerol saline: Used to transport feces from patients suspected to be suffering from bacillary dysentery.
- Viral Transport Medium: Viral Transport Medium (VTM) is ideal for the diagnosis of viral infection. Ocular, respiratory, and tissue swabs can be submitted in this medium. Fluid samples such as tracheal wash specimens or peritoneal fluid should be submitted as is, in sterile vials which prevent desiccation. In the absence of viral transport medium, submit swabs in sterile, sealed vials with several drops of saline added, to prevent desiccation. Cotton, plastic, wood-handled, and dacron and other synthetic swabs are all acceptable. Calcium alginate swabs should be avoided. Bacterial transport media are not appropriate for virology.
- Anaerobic Transport Medium (ATM): is a mineral salt base semi-solid media with reducing agents designed as a holding medium for maintaining the viability of anaerobic bacteria It contains buffered mineral salts in a semi-solid media with sodium thioglycolate and cysteine added to provide a reduced environment. Resazurin may also be added as a redox indicator to reveal exposure to oxygen by turning pink. It provides an environment, which maintains the viability of most microorganisms without significant multiplication and allows for dilution of inhibitors present in clinical material. Examples include thioglycollate broth
There are several types of bacterial culture methods that are selected based on the agent being cultured and the downstream use.
Broth cultures Edit
One method of bacterial culture is liquid culture, in which the desired bacteria are suspended in a liquid nutrient medium, such as Luria Broth, in an upright flask. This allows a scientist to grow up large amounts of bacteria for a variety of downstream applications.
Liquid cultures are ideal for preparation of an antimicrobial assay in which the experimenter inoculates liquid broth with bacteria and lets it grow overnight (they may use a shaker for uniform growth). Then they would take aliquots of the sample to test for the antimicrobial activity of a specific drug or protein (antimicrobial peptides).
As an alternative, the microbiologist may decide to use static liquid cultures. These cultures are not shaken and they provide the microbes with an oxygen gradient. 
Agar plates Edit
Microbiological cultures can be grown in petri dishes of differing sizes that have a thin layer of agar-based growth medium. Once the growth medium in the petri dish is inoculated with the desired bacteria, the plates are incubated at the optimal temperature for the growing of the selected bacteria (for example, usually at 37 degrees Celsius, or the human body temperature, for cultures from humans or animals, or lower for environmental cultures). After the desired level of growth is achieved, agar plates can be stored upside down in a refrigerator for an extended period of time to keep bacteria for future experiments.
There are a variety of additives that can be added to agar before it is poured into a plate and allowed to solidify. Some types of bacteria can only grow in the presence of certain additives. This can also be used when creating engineered strains of bacteria that contain an antibiotic-resistance gene. When the selected antibiotic is added to the agar, only bacterial cells containing the gene insert conferring resistance will be able to grow. This allows the researcher to select only the colonies that were successfully transformed.
Agar based dipsticks Edit
Miniaturised version of agar plates implemented to dipstick formats, eg. Dip Slide, Digital Dipstick  show potential to be used at the point-of-care for diagnosis purposes. They have advantages over agar plates since they are cost effective and their operation does not require expertise or laboratory environment, which enable them to be used at the point-of-care.
Stab cultures Edit
Stab cultures are similar to agar plates, but are formed by solid agar in a test tube. Bacteria is introduced via an inoculation needle or a pipette tip being stabbed into the center of the agar. Bacteria grow in the punctured area.  Stab cultures are most commonly used for short-term storage or shipment of cultures.
Culture collections Edit
Microbial culture collections focus on the acquisition, authentication, production, preservation, catalogueing and distribution of viable cultures of standard reference microorganisms, cell lines and other materials for research in microbial systematics.   Culture collection are also repositories of type strains.
Major national culture collections.  
Collection Acronym Name Location ATCC American Type Culture Collection Manassas, Virginia BCCM Belgian Co-ordinated Collections of Micro-organisms Decentralized, Coordination Cell in Brussels, Belgium CCUG Culture Collection University of Gothenburg Gothenburg, Sweden CECT Colección Española de Cultivos Tipo Valencia, Spain CIP Collection d'Institut Pasteur Paris, France DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen Braunschweig, Germany ICMP International Collection of Microorganisms from Plants Auckland, New Zealand JCM Japan Collection of Microorganisms Tsukuba, Ibaraki, Japan NCTC National Collection of Type Cultures Public Health England, London, United Kingdom NCIMB National Collection of Industrial, Food and Marine Bacteria Aberdeen, Scotland
Solid plate culture of thermophilic microorganisms Edit
For solid plate cultures of thermophilic microorganisms such as Bacillus acidocaldarius, Bacillus stearothermophilus, Thermus aquaticus and Thermus thermophilus etc. growing at temperatures of 50 to 70 degrees C, low acyl clarified gellan gum has been proven to be the preferred gelling agent comparing to agar for the counting or isolation or both of the above thermophilic bacteria. 
Virus or phage cultures require host cells in which the virus or phage multiply. For bacteriophages, cultures are grown by infecting bacterial cells. The phage can then be isolated from the resulting plaques in a lawn of bacteria on a plate. Virus cultures are obtained from their appropriate eukaryotic host cells. The streak plate method is a way to physically separate the microbial population, and is done by spreading the inoculate back and forth with an inoculating loop over the solid agar plate. Upon incubation, colonies will arise and single cells will have been isolated from the biomass. Once a microorganism has been isolated in pure culture, it is necessary to preserve it in a viable state for further study and use. Stock cultures have to be maintained, such that there is no loss of their biological, immunological and cultural characters.
Isolation of pure cultures Edit
For single-celled eukaryotes, such as yeast, the isolation of pure cultures uses the same techniques as for bacterial cultures. Pure cultures of multicellular organisms are often more easily isolated by simply picking out a single individual to initiate a culture. This is a useful technique for pure culture of fungi, multicellular algae, and small metazoa, for example.
Developing pure culture techniques is crucial to the observation of the specimen in question. The most common method to isolate individual cells and produce a pure culture is to prepare a streak plate. The streak plate method is a way to physically separate the microbial population, and is done by spreading the inoculate back and forth with an inoculating loop over the solid agar plate. Upon incubation, colonies will arise and single cells will have been isolated from the biomass. Once a microorganism has been isolated in pure culture, it is necessary to preserve it in a viable state for further study and use. Stock cultures have to be maintained, such that there is no loss of their biological, immunological and cultural characters.
- plate count: A means to identify the number of actively growing cells in a sample.
Figure: Selective media can be used to restrict the growth of non-target bacteria.: Urine cultured on Oxoid Brilliance UTI Agar plate. 1uL of urine spread onto the agar surface. The top sample is from patient with clinical urinary tract infection (UTI). The bottom sample is a mixed culture.
There are a variety of ways to enumerate the number of bacteria in a sample. A viable cell count allows one to identify the number of actively growing/dividing cells in a sample. The plate count method or spread plate relies on bacteria growing a colony on a nutrient medium. The colony becomes visible to the naked eye and the number of colonies on a plate can be counted. To be effective, the dilution of the original sample must be arranged so that on average between 30 and 300 colonies of the target bacterium are grown. Fewer than 30 colonies makes the interpretation statistically unsound and greater than 300 colonies often results in overlapping colonies and imprecision in the count. To ensure that an appropriate number of colonies will be generated several dilutions are normally cultured. The laboratory procedure involves making serial dilutions of the sample (1:10, 1:100, 1:1000 etc. ) in sterile water and cultivating these on nutrient agar in a dish that is sealed and incubated. Typical media include Plate count agar for a general count or MacConkey agar to count gram-negative bacteria such as E. coli. Typically one set of plates is incubated at 22°C and for 24 hours and a second set at 37°C for 24 hours. The composition of the nutrient usually includes reagents that resist the growth of non-target organisms and make the target organism easily identified, often by a color change in the medium. Some recent methods include a fluorescent agent so that counting of the colonies can be automated. At the end of the incubation period the colonies are counted by eye, a procedure that takes a few moments and does not require a microscope as the colonies are typically a few millimeters across.
The pour plate method is used when the analysis is looking for bacterial species that grow poorly in air. The initial analysis is done by mixing serial dilutions of the sample in liquid nutrient agar which is then poured into bottles. The bottles are then sealed and laid on their sides to produce a sloping agar surface. Colonies that develop in the body of the medium can be counted by eye after incubation. The total number of colonies is referred to as the Total Viable Count (TVC). The unit of measurement is cfu/ml (or colony forming units per milliliter) and relates to the original sample. Calculation of this is a multiple of the counted number of colonies multiplied by the dilution used. Examples of a viable cell count are spread plates from a serial dilution of a liquid culture and pour plates. With a spread plate one makes serial dilutions in liquid media and then spreads a known volume from the last tube in the dilution series. The colonies on the plate can then be counted and the concentration of bacteria in the original culture can be calculated. In the pour plate method a diluted bacterial sample is mixed with melted agar and then that mixture is poured into a petri dish. Again the colonies would be counted and the viable cell count calculated.
Growth of Bacteria (With Diagram) | Microbiology
In this article we will discuss about:- 1. Definition of Growth 2. Measurement of Bacterial Growth 3. Multiplication of Unicellular Bacteria 4. Determination of Generation Time 5. Growth Curve 6. Continuous Culture 7. Synchronous Culture 8. Culture Media 9. Enrichment Culture 10. Requirements of Macro- and Micro-Elements for Growth 11. Physical Factors Influencing Growth.
- Definition of Growth
- Measurement of Bacterial Growth
- Multiplication of Unicellular Bacteria
- Determination of Generation Time
- Growth Curve
- Continuous Culture
- Synchronous Culture
- Culture Media
- Enrichment Culture
- Requirements of Macro- and Micro-Elements for Growth
- Physical Factors Influencing Growth
1. Definition of Growth:
In biology, growth is generally defined as an irreversible increase in cellular mass due to active synthesis of all the constituents. Growth results in increase of cell number (except in coenocytic organisms). In multicellular organisms, this increase in cell number leads to increase in size, because the daughter cells remain together.
In contrast, in unicellular organisms, cell multiplication leads to increase in number of individuals in a population, i.e. the population size. So, for bacteria, majority of them being unicellular organisms, growth may be defined as the increase in number of cells in a population. It should be remembered, however, that in bacteria and other unicellular organisms, too, a young daughter cell grows in size before it attains a stage when it can divide to complete a cell-cycle.
2. Measurement of Bacterial Growth:
Since bacterial growth results in increase in number and, therefore, in population size, several alternatives are available for its measurement. The cell density i.e. number of cells per unit volume of medium may be determined by counting, by measuring the optical density, by estimating the dry weight or protein, etc. Some of these are direct methods and some are indirect.
One of the obvious ways for measurement of growth of a growing population in culture (cultivation of organisms in the laboratory on media in which they can grow) is their direct counting under the microscope. The number of cells in a given volume of the culture can be counted using specially ruled grooved slides similar to those used by medical technicians for counting blood cells.
The grooved portion of the slide has a known depth and area which is divided into several equal squares. These slides are called counting chambers, different types of which are available, like Petroff-Hausser, Neubauer, Thoma etc. (Fig. 7.1).
A drop of the bacterial suspension is placed on the groove of the slide, covered with a cover slip, lightly pressed to remove excess fluid and the slide is examined under the high-power objective of a phase-contrast microscope. The number of bacteria per small square is counted for fairly good number of squares, averaged, and from the mean the total number of bacteria per ml of the suspension is calculated.
In Petroff-Hausser counting chamber an area of 1 mm 2 is divided into 25 equal squares and the depth of the groove is 0.02 mm. If the number of bacteria is too high for counting, the original culture may be suitably diluted and for calculating the final count the dilution factor is taken into account. Motile bacteria are to be immobilized before transferring to the counting chamber.
The number of bacteria obtained by the above procedure gives the total count. It is obvious that the total count includes both living as well as dead cells. Living or viable bacteria are capable of producing progeny cells by division.
On a solidified nutrient medium a viable bacterium divides and re-divides to form a large number of progeny cells to form a colony. So, the colony-forming ability is a test for viability. A dead or non-viable bacterium is unable to form a colony under any condition of growth. The number of living bacteria per unit volume of a culture or a population is known as its viable count.
The method followed for determining viable count is based on the principle of serial dilution, first developed by Joseph Lister and later perfected by Robert Koch. There are two variations of the dilution plating technique — the spread plate and the pour-plate methods. In the first method, a measured quantity of a serially diluted sample of the original culture is spread evenly on the surface of the solidified growth medium.
On incubation at an optimum temperature, each viable bacterial cell forms a discrete colony on the surface of the medium. Assuming that each visible colony is the progeny of a single bacterium, the total number of colonies is taken as the number of viable cells present in the quantity of the diluted sample spread on the medium. So, by counting the number of colonies, the viable count can be easily calculated. For reliable results, a good number of replications are necessary.
The method is diagrammatically shown in Fig. 7.2:
[One ml of the bacterial culture containing
10 7 — 10 9 cells/ml is transferred aseptically with a sterilized pipette to 9 ml of sterilized water or normal saline, mixed thoroughly to get a uniform suspension. Then 1 ml of the 1:10 dilution of the original culture is transferred with a fresh sterile pipette to another tube containing 9 ml of water or normal saline to obtain 1:100 (10 2 ) dilution.
The process is continued till a dilution of 10 -7 is reached. Next 0.1 ml aliquots of the final or last two dilutions are uniformly spread with a sterile bent glass rod over the surface of petridishes containing suitable agar medium. On incubation, colonies of bacteria appear on the plates. Number of colonies on replicate plates is counted, their mean determined and the viable count is calculated, taking into consideration the dilution factor. In the example shown in the figure, the viable count comes to 30.7 x 10 8 = 3.07 x 10 9 /ml.]
For making counting of colonies easier, an instrument called a colony-counter may be used. Essentially, it is a box with a slanting top containing a circular hole of 10-12 cm diameter covered with a frosted glass. There is arrangement for illumination inside the box.
For facilitating counting of small colonies, a magnifying glass of about the same diameter as that of the hole is clamped on a bracket. For counting the petridish is placed in an inverted position on the frosted glass, illuminated from below and observed through the magnifying glass. A marker pen may be used to record the colonies while counting, so that the same colony is not counted more than once.
The second variation of the serial dilution method, the pour-plate technique, is essentially similar to the dilution plating technique, except that the diluted cell suspension is mixed with molten agar medium just before pouring in plates. The temperature should not be so high as to kill bacteria and not so low that agar begins to solidify.
Good quality agar sets at a temperature of about 42°C, so 45°-50°C is ideal for pouring. On incubation, colonies appear first on the surface. Gradually, bacteria caught inside the agar also form colonies. These colonies are at first lens-shaped before they emerge on the surface. The number of colonies can be counted as in case of spread plates.
The above two methods can be applied for counting aerobic bacteria only, but not for anaerobic bacteria which are unable to grow under normal oxygen concentration of air. For such organisms the dilution plates have to be incubated under oxygen-free atmosphere.
Common vacuum desiccators in which air is replaced by an inert gas like nitrogen can be used. For highly oxygen-sensitive anaerobes, an oxygen-absorbent like alkaline pyrogallol may have to be additionally used. Also, various types of anaerobic jars are commercially available for this purpose.
A different method for determining viable count is the membrane-filter technique. It is mostly used in case of naturally occurring microbial populations, where the cell density is not high (generally -10 6 cells/ml), such as for counting coliform bacteria in water samples.
A measured volume of the sample is passed through a sterile membrane filter under negative pressure. The filter should have a pore size less than the diameter of average bacterial cells (less than 1μm). After filtration, the membrane filter disc is aseptically transferred on the surface of suitable sterilized agar medium and incubated till visible colonies appear on the filter surface. The colonies are then counted and the viable count per unit volume of the sample is calculated in the usual way.
An entirely different procedure based on statistical principle is the determination of the most probable number (MPN) of a specific group of microorganisms in natural population. The procedure is generally applied in bacteriological analysis of water samples, but it may be also used for other groups of naturally occurring organisms.
For determination of the MPN of coliform bacteria in a sample of water, aliquots of 10 ml, 1 ml and 0.1 ml of the water sample are inoculated into 5 replicate tubes of lactose broth, each containing an inverted Durham’s tube.
Coliform bacteria ferment lactose to produce gas which collects in the Durham’s tubes.
After incubation for 48 h at 32°C, the tubes are examined for presence of gas, and the number of tubes showing gas accumulation is counted (Fig. 7.3):
The most probable number of coliform bacteria in the water sample is then determined from a standard MPN table (Table 7.1):
While counting bacteria in a population is one way for determination of growth, another parameter — viz. measurement of bacterial mass — can also be utilized. Gravimetric estimation of fresh bacterial cells or of dry cells provides a direct method.
Fresh weight of a bacterial mass can be obtained by collecting the cells through centrifugation, washing them with distilled water to remove the soluble ingredients of the culture medium and transferring the pellet to a pre-weighed container. For determination of dry weight, the washed cell mass is kept at 110°C overnight and weighed. Very careful handling is essential for obtaining accurate result, since the quantity is very small for laboratory-scale cultures.
Other methods are also available for measurement of bacterial mass, though they are somewhat indirect. One of these is estimation of total nitrogen of the cell mass by the micro-kjeldahl method. Total nitrogen content of an actively growing culture increases linearly with cell mass.
A similar relationship also exists between total carbon and cell weight. Total carbon content can be estimated by the Van Slyke-Folch method. For routine purpose, estimation of total protein of an aliquot of the culture by any of the common methods gives satisfactory and reliable results.
For total protein, the cells are lysed by treatment with alkali resulting in release of the cell contents, followed by centrifugation to eliminate the cell debris and treating an aliquot with a colouring reagent, like biuret, Folin, Coomassie blue etc.
The colour developed is then measured in a colorimeter. From the colorimeter reading, the quantity of protein (mg/ml) is then read off from a standard curve. A standard curve is prepared using an authentic protein like bovine serum albumin and the same coloring reagent. A standard curve shows known quantities of the protein in one axis and the corresponding colorimetric readings in the other axis.
An indirect but rapid and handy method of estimating cell mass is turbidemetry. Cell mass which is directly proportional to the population density can be accurately measured by this method. As the cell number increases with growth, a bacteriological culture medium turns more and more turbid, allowing less and less light to pass through and at the same time scattering more and more of light by the suspended cells.
For most common bacteria, visible turbidity appears when the cell density is between 10 6 to 10 1 cells/ml. When the culture contains less cells, turbidimetric measurement of growth is not feasible. The instrument used for this method is called a photo-electric colorimeter which measures the optical density of a suspension or also the colour density of a coloured solution.
The optical density of a bacterial suspension is measured by transferring a portion of a liquid culture to a colorimetric glass tube usually having a diameter (light-path) of 1 cm against a blank (control) which is usually the uninoculated culture medium. A suitable light filter (usually green or blue) is used. Optical density can be measured either in terms of absorbance (A) or transmission (T%).
Generally, a colorimeter has both types of scales. Absorbance is the logarithm of the ratio of intensities of incident light (I0) and transmitted light (1) i.e. A = log (I0/I). It is given in log scale. Transmission percent is the percentage of incident light passing through the suspension and is calibrated in an arithmetic scale. The two scales run obviously antiparallel e.g. the blank has an absorbance of O and T% of 100.
As turbidity increases absorbance increases and transmission % decreases. The colorimeter is provided with a light source, some filters for selecting light of a desired range of wave lengths, a colorimeter tube with known light-path, a photo-cell which converts incident light to electric current, a device for amplification of the current so produced and finally a galvanometer for measurement of the electric current (Fig. 7.4). A linear relationship between bacterial cell density and absorbance or transmission exists only when the suspension is relatively thin (Fig. 7.5).
Another instrument known as a nephelometer has more or less the same components as a colorimeter, but is more sensitive. It measures the light scattered by the bacterial cells in suspension. The higher the cell density more is the quantity of scattered light.
The apparatus is so designed that it can capture the light rays scattered by the suspended bacteria at right angles to the beam of incident light and convert them into electricity which can then be measured in the usual way.
Both colorimetry and nephelemetry can be used for measurement of growth of only unicellular organisms which form a stable, uniform suspension. If colorimeter reading or nephelometer reading is calibrated against cell number or cell mass, these methods provide reliable means for estimation of growth.
3. Multiplication of Unicellular Bacteria:
Majority of bacteria are unicellular organisms and most of them multiply by binary fission which means that each bacterium divides to produce two identical cells. Each of them, after attaining maturity, undergoes similar binary fission to produce two daughter cells. Thus, under ideal conditions, cell number as well as mass double itself after given time intervals depending on the species and on the growth conditions.
The time interval between two successive divisions is called the generation time or doubling time. After each unit generation time the number of bacteria doubles. Thus, under optimal conditions, growth of bacteria takes place by geometric progression with a constant factor of 2. Starting from a single bacterium, the increase in number can be represented as 2 0 —>2 1 —>2 2 —>2 3 —>2 4 —>2 5 —>….—>2 n after n divisions.
4. Determination of Generation Time:
If the number of cells per unit volume of an actively growing bacterial culture at a time to is taken as N0, then the number at time t during which n divisions have taken place is given by the equation:
Nt = N0.2 n , where N, represents the number of bacteria at time t.
Expressed logarithmically, the above equation becomes:
It is seen from the last equation that by counting the number of bacteria present in an actively growing culture at time t0 and t, it is possible to determine the number of divisions that has occurred during the time interval between t0 and t.
Once this is done, the generation time (g) – which is the time between two successive divisions – can be easily determined, because g = t/n. Again, the number of divisions per hour which is known as the doubling rate (v), becomes v = n/t and also v = 1/g. An example may be given for clarification of the above mathematical considerations.
If an actively growing culture has a cell count of 10 4 /ml at a certain time and which increases to 10 8 /ml after a lapse of 6 hours, then the organism has a doubling rate (v):
And the generation time (g) is
The generation time varies from one species to another and it also depends on the growth conditions. Under optimal conditions, Escherichia coli has a generation time of 20 min which means a single E. coli cell can produce 1,024 cells after 200 min during which 10 divisional cycles have taken place.
Exponential or Logarithmic Growth:
When, in a bacterial culture, the population doubles at each generation, the growth is said to be exponential or logarithmic, because the population increases as an exponent (power) of 2 and log2 of the number of cells increases in direct proportion to time. A semi-logarithmic plot of the cell number per unit volume of an exponentially growing population against time gives a straight line (Fig. 7.6).
The linear relation between logarithm of cell number and time holds good, only when all the cells in a growing culture are viable. In practice, such an ideal behaviour is hardly expected, because some cells lag behind or become non-viable.
5. Growth Curve:
Bacterial growth in a flask — or any other container which can be as big as an industrial fermenter — holding a growth supporting medium is known as a batch culture. The batch culture represents a “closed” system, because the nutrients initially present are gradually consumed during growth-producing metabolic end-products which accumulate in the culture vessel causing change of pH value. There is no provision for addition of fresh nutrients or for removal of end products or adjustment of the pH value which also changes during growth.
When the logarithm of the number of bacteria per unit volume of such a batch culture is plotted against time (hr) beginning with the transfer of some viable organisms to the culture vessel (inoculation) a sigmoid growth curve is obtained (Fig. 7.7).
A typical growth curve shows several distinct phases. These are known as the lag phase, the exponential or logarithmic phase, the stationary phase and the death phase. Sometimes, the later part of lag phase is called the acceleration phase and the early part of the stationary phase is called deceleration phase.
The lag phase represents the time interval between inoculation and the onset of the exponential growth. During the lag phase, the cell number does not increase, but individual bacteria grow in size due to active synthesis of cellular ingredients like protein, nucleic acids and carbohydrate.
During the later part of this phase, some of the bacteria begin to divide resulting in a slow rise in total count (acceleration phase). There may be several reasons why the bacteria do not start growing immediately after inoculation in a fresh medium.
If the inoculum is taken from an old culture, or from a culture which was growing in a medium having a different composition, the inoculated cells require some time to adapt themselves in the new environment. So, the lag phase may be considered as an adaptation phase during which the inoculated cells do not remain idle, but they are engaged in synthesizing cell materials as a preparation to initiate active growth.
From the lag phase, the bacteria pass into the exponential phase (logarithmic phase) through the intermediate acceleration phase and they are now fully equipped to initiate growth at a maximum rate. Most cells of the population divide regularly at an interval of each unit generation time resulting in exponential increase of cell number and mass.
However, all cells of the total population do not divide simultaneously, rather they divide asynchronously. As a result, the cell number does not increase in a step-wise manner. The maximal growth rate is maintained throughout the exponential phase and it continues till a point is reached where the population density becomes so high that the growth medium is unable to support further growth.
The growth rate falls and ultimately stops. At this point, the culture enters into the stationary phase. Duration of the exponential phase depends not only on availability of nutrients, but also on other factors, like oxygen supply, pH, temperature etc. and, naturally, also on the generation time. The logarithmic (log) phase cells exhibit their highest activity and, therefore, they are most suitable for measurement of generation time, various biochemical properties and cell size.
As the culture enters into the stationary phase, there is no net increase in cell number, although a majority of the bacteria still remains in a viable state and they continue life activities at the cost of reserve substances stored in the cells.
The duration of this phase is highly variable among different species and may be few hours to several days. From practical point of view, the stationary phase cells are of special significance, because many secondary metabolic products, for example antibiotics, are produced in this phase. Also, for organisms which are cultivated as source of biomass, harvesting in this phase gives maximum yield.
The stationary phase is succeeded by the death phase during which the viable cell count falls exponentially, although total cell count may continue unchanged for quite some time. Thereafter, the total count also falls indicating that the cells undergo lysis and they disappear. Lysis may be due to the activity of their own enzymes (autolysis). It is possible that the cell materials released by lysis may provide nutrition to the still living cells for some time. However, the exact causes of death of bacteria are not clearly understood.
An important parameter of growth is the yield which can be determined from the growth curve of a batch culture by measuring the dry weight of the total population at the beginning and at the end of the exponential phase. Thus, if Mmax denotes bacterial mass (dry weight, g) at the end of the exponential phase, and M0 the initial mass, then yield M is, M = Mmax – M0 (Fig. 7.8).
Usually, yield is expressed in relation to the substrate consumption as yield coefficient (Y) which is the ratio of yield (dry wt. g) and the quantity (g) of substrate utilized. Conventionally, yield coefficient per mole of substrate consumed is called molar yield coefficient (Ym).
Another form of expression of the yield coefficient is the energy yield coefficient (YATP) which is yield per mole of ATP consumed. For calculating this parameter it is essential to know the pathway of carbohydrate (energy source) breakdown of the particular organism and the quantity (moles) of ATP produced per mole of the carbohydrate.
For example, Streptococcus faecalis or Saccharomyces cerevisae breaks down glucose by the EMP (Embden-Meyerhof-Parnas pathway) and, when growing in absence of air, produces 2 moles of ATP/mole of glucose.
While molar yield coefficient (Ym) for both these organisms is 20, i.e. both produce 20 g dry cells per mole of glucose (180 g), energy yield coefficient (YATP) for both is 10. Under aerobic conditions, the yield increases significantly, because of higher energy production by complete oxidation of the substrate.
6. Continuous Culture:
The fate of a microbial population growing in a “closed” system like a batch culture is comparable to the fate of a multicellular organism having birth, growth, maturity, and death. The environment in a batch culture is subject to constant changes due to continuous depletion of nutrients, shifting of pH value, partial pressure of dissolved gases, accumulation of toxic metabolites etc.
Due to such changes, the duration of active growth phase is also limited. For many critical experiments, it becomes necessary to maintain a culture in an active state of growth over a prolonged time. This can be achieved by a continuous culture which provides for growth of an organism in a constant environment. A continuous culture represents an “open” system in contrast to a batch culture.
One of the devices for growing microorganisms in a continuous culture is a chemostat developed by Novick and Szilard (1950). In its simplest form, a chemostat consists of a culture vessel provided with an inlet for regulated entry of fresh culture medium, an arrangement for pumping in sterilized air, and an Outlet for maintaining a constant volume of liquid in the culture vessel (Fig. 7.9).
The arrangements in a chemostat ensure a constant regulated flow of fresh medium into the culture vessel and simultaneous removal of equal quantity of culture fluid from the vessel through an outlet device. As a result, a constant volume is always maintained in the vessel.
The system is also provided with an arrangement for pumping in sterilized air through the culture to maintain adequate aeration. In contrast to a batch culture, a continuous culture is more amenable to control. On one hand, the continuous inflow of fresh medium-prevents depletion of essential nutrients and, on the other hand, continuous outflow removes spent medium containing toxic metabolites and bacterial cells. These arrangements ensure a prolonged growth in a chemostat.
The importance of a chemostat culture lies in the fact that the growth rate of an organism can be controlled. This can be achieved by supplying one of the essential nutrients, like carbon and energy source, at a sub-optimal concentration in the inflowing medium.
As a result, the organisms present in the culture are starved and are unable to grow at maximum growth rate. At the same time, the inflow rate can be so adjusted that the particular growth-limiting nutrient is immediately and fully consumed by the hungry organisms. The outflow removes continuously a fraction of the population to maintain a constant cell density.
Thus, it becomes possible to establish a steady-state of growth. In a steady-state, the growth rate is constant and it equals the rate of inflow of medium which is known as the dilution rate. The dilution rate is so adjusted that the concentration of the critical nutrient in the chemostat is practically nil, because the nutrient is instantaneously consumed by the starving population.
As a result, the growth rate in the chemostat is maintained at a sub-maximal value. Thus, by regulating the dilution rate, it is practically possible to maintain a steady-state culture almost indefinitely which is in sharp contrast to a batch-culture which passes through different growth phases — ultimately terminating in death.
The relation between substrate concentration and growth rate is closely comparable with the relation between substrate concentration and velocity of enzyme reaction as depicted by the famous Michaelis-Menten equation. The growth rate (μ) increases linearly with substrate (growth limiting nutrient) concentration [s] up to a certain point, and then declines to become flat. The substrate concentration at which the growth rate is half of the maximum rate of growth is designated by Ks (Fig. 7.10).
7. Synchronous Culture:
A synchronous culture is one in which all the cells of a particular population are in the same stage of development and they divide simultaneously. In a common batch-culture, the population contains cells in all possible stages of development, some have just been produced by fission, others are in intermediate stages and still others are mature and ready for division.
All these stages are parts of the cell-cycle. For this reason, the cell population of a batch culture divides asynchronously producing a typical exponential growth curve indicating that the logarithms of cell number as well as of cell-mass increase linearly with time (see Fig. 7.6).
In a synchronous culture, on the other hand, one would expect a step-wise increase in cell number, because there would be no increase in cell number between two successive divisions. But the cell mass would still show a linear increase with time, similar to that found in a batch culture. This is due to the fact that the newly born cells go on increasing in mass between two successive divisions, though their number does not increase (Fig. 7.11).
Synchronization of cell division in a bacterial population can be achieved in several ways. One of them is repeated alternation of the incubation temperature. A culture growing at the optimal temperature is exposed to a lower temperature, so that the growth is slowed down considerably. After some time, the culture is again brought to its optimal temperature.
The time interval is adjusted according to the growth rate (generation time) of the organism. By lowering the temperature, cell division is delayed and all cells divide simultaneously when the temperature is raised to the optimal level. The treatment has to be continued through several cycles to obtain satisfactory results. Another method of getting a synchronously dividing population of cells is by filtration through a membrane filter.
An asynchronous population of bacteria is filtered whereby the cells are adsorbed in the pores of the membrane filter. In the adsorbed state the cells continue dividing producing progeny cells which are not adsorbed. A reverse flow of sterilized medium through the membrane filter washes away the newly born progeny cells into the filtrate. Thus, a homogeneous population of cells which are approximately at the same stage of development can be obtained. Such cells divide synchronously for a few generations.
The utility of a synchronous culture lies in the fact that the whole culture can be taken as a multicellular complex consisting of individuals having identical development. In contrast, an asynchronous culture can give only an average picture about individual cells.
Sometimes, it becomes necessary to gain insight into the sequence of events taking place in individual cells. For smallness of size, study of such events in single bacterial cells is not feasible. A synchronous culture provides this opportunity, because all cells are in the same stage of development. For example, one might want to study the replication of DNA in relation to the cell cycle.
Samples withdrawn at suitable intervals during one unit generation time of a synchronous culture would yield cells at different stages of the cell cycle and each sample would contain cells of a particular stage. A measurement of incorporation of a radioactive DNA precursor would provide useful information of DNA synthesis at different phases of the growth cycle.
8. Culture Media:
Majority of bacteria and many eukaryotic microorganisms like algae and fungi can be cultivated under artificial conditions (as opposed to their natural habitats) on suitable culture media. A culture medium must contain all the raw materials that are needed by the particular organism to build up its cellular constituents — carbohydrates, proteins, lipids, nucleic acids etc.
Since water is the most important constituent of all living systems, microorganisms can best thrive in an aqueous medium. The main constituent of the culture medium is, therefore, water, in which the other ingredients are present in a dissolved state.
Microorganisms, like plant cells, are generally invested with a cell wall and they can take up nutrients only when they are in a dissolved state. The major barrier for entry of nutrients into the cell is, however, not the cell wall, but the cytoplasmic membrane. Some of the soluble nutrients can diffuse passively through the membrane, but for the majority there are specific transport systems located in the membrane which help in the uptake of nutrients from the culture medium.
The major elements that are essential for growth of all microorganisms are carbon, nitrogen, hydrogen, oxygen, phosphorus, sulfur, magnesium, calcium, potassium and iron. Many microorganisms also require traces of one or more minor elements, like manganese, molybdenum, zinc, cobalt, nickel, copper, boron, sodium and silicon.
All the elements required for growth have to be provided in the culture medium. Majority of bacteria and all fungi have a heterotrophic mode of nutrition and they are dependent on one or the other organic compound as source of carbon and energy. In general, microorganisms can take up all the other elements in inorganic form.
Thus, for growing the common bacteria and fungi present in soil or water, an inorganic salts medium containing a single organic compound proves adequate. But pathogenic bacteria often refuse to grow in such simple culture media and require supplementation of complex organic compounds, probably because they get accustomed to such compounds while growing in the body of the host. For example, Haemophilus requires haemin and nicotinamide adenine dinucleotide (NAD) in their growth medium.
Although common microorganisms (bacteria and fungi) can grow in an inorganic salt medium with a single carbon-source (generally glucose), they grow much faster when the medium is enriched by addition of some complex organic compounds.
On this basis, culture media can be distinguished into two types:
The complex media contain one or more substances of undefined chemical composition. Commonly employed substances of this type are beef-extract, peptone, tryptone, yeast-extract, malt extract, blood, serum, egg albumin, potato extract, straw-infusion etc.
Casein-hydrolysate is another complex supplement, though its composition is more or less known. A complex bacteriological medium very commonly used for obtaining rapid and good growth is nutrient broth which contains beef-extract, peptone and sodium chloride.
Similarly, for growing saprophytic fungi, a common complex medium is malt-extract agar. Potato-dextrose agar — which contains extract of boiled potato, glucose and some salts — is another medium for growing fungi. Straw-infusion broth is a good complex medium for growth of soil actinomycetes. For growth of pathogenic bacteria blood agar, serum-albumin agar are often used. Among complex media are also to be counted some natural substances like milk, potato, carrot etc.
The chemically defined media, also called synthetic media, contain substances of known chemical composition and each of them is present in a known concentration. A simple synthetic medium which supports growth of common bacteria including Escherichia coli may be prepared from the following ingredients: K2HPO4 7.0, KH2PO4 3.0, Na3-citrate. 3H2O 0.5, MgSO4. 7H20 0.1, (NH4)2SO4 1.0, glucose 2.0, FeSO4 . 7H20 0.01, CaCl2 .2H20 0.01 (g/1). Similarly, for fungi, a synthetic medium is Czapek-Dox.
Culture media can be used in liquid form (broth) or in the form of a soft or a relatively hard gel (solid media). Gelatin which was used in the early days of microbiology as gelling agent is no longer in use except for special purposes. It has been completely replaced by agar-agar which is a much superior agent.
Agar-agar is a complex, highly cross-linked polysaccharide extracted from some marine red algae. At a concentration of 1.5-2% (w/v) it produces a solid gel, and at half the above concentration a soft gel. It sets to a gel at 42°-45°C and the gel can be melted at 100°C. The process of gelling and melting can be repeated several times, unless the pH of the medium is acidic.
Unlike gelatin, agar is not hydrolysed by most bacteria. The surface of agar media remains more or less dry, so that colonies appearing on the surface do not spread, and discrete colonies can be easily picked up. All these qualities make agar-agar an ideal agent for solidification of microbiological culture media.
There are some organisms, though very few, like Nitrosomonas which refuse to grow on agar media. For such highly fastidious organisms, the use of an inorganic gel becomes necessary, such as silica gel. It can be prepared by acidifying a solution of sodium silicate with hydrochloric acid. After setting, the gel is washed free of sodium chloride and excess acid. After drying the gel is allowed to absorb sterilized culture medium before inoculation.
When a mixed population of different types of microorganisms is inoculated in a culture medium which allows selective growth of a single type or of a specific group, the medium is considered as selective. Selective media have to be designed according to the necessity for artificially increasing the number of a specific organism or, more commonly, for a specific group of organisms originally present in a mixed population (enrichment) or for direct isolation of a particular type from a mixed population.
A few examples of selective media can be cited. If one intends to study the types of nitrogen- fixing bacteria present in a certain soil, selective medium would contain other ingredients except any nitrogenous compound. In such a medium, organisms which are capable of utilizing atmospheric nitrogen alone would grow.
Again, if one wants to find out the number and select antibiotic resistant strains in a large population, the medium used for the purpose would contain the particular antibiotic at a concentration which is inhibitory for the sensitive strains. The nitrogen-free medium in the first case and the antibiotic-containing medium are examples of selective media.
Whereas a selective medium permits the growth of a selective organism or a selective group of organisms, a differential medium allows growth of various types of organisms present in a mixed population, but at the same time helps to differentiate a particular organism or a group of organisms from the rest.
Thus in a mixed population, the presence of a particular type can be detected. For example, the presence of coliform bacteria in a water sample suspected to be contaminated with fecal matter can be detected by gas production from lactose. The ability to utilize a certain sugar can be similarly tested on a solid medium containing the particular sugar and a mixture of indicator dyes — eosine and methylene blue.
The organisms capable of fermenting the sugar to produce acid form colonies which absorb the dyes and produce a metallic sheen. Thus, on this differential medium (eosine-emthylene blue agar, EMB) the organisms can be visually differentiated from the rest. The endo-agar medium prepared by adding decolorized basic fuchsin in a lactose containing agar medium is another differential medium for identification of coliform bacteria.
9. Enrichment Culture:
Enrichment culture technique, developed by Winogradsky and Beijerinck, is based on the Darwinian principle of the survival of the fittest. In an enrichment culture, the environment is preset in such a manner that an organism or a group is selectively encouraged to grow and multiply, so that in a mixed population they become predominant.
The environmental factors that can be utilized for such selective growth or enrichment include carbon, nitrogen and energy sources, oxygen tension, pH, temperature, light etc. These factors have to be selected on the basis of the knowledge about the physiological-biochemical abilities of the organism desired to be enriched.
The question of enrichment arises when the desired organism needs to be isolated from a mixed population in which it forms a minority and is far outnumbered by other organisms. The usual dilution plating method cannot be employed as such, because during dilution the desired organisms are eliminated.
Therefore, before making dilution plates, the number of the desired organisms has to be increased preferentially over the non-desired organisms. In an enrichment culture the competition between the desired and non-desired organisms is removed or minimized, so that after few passages through enrichment culture, the desired organisms become the majority and then they can be isolated by the usual dilution procedure.
Success in such enrichment procedure naturally depends on how effective the selective conditions are. For creating such selective conditions, the knowledge about the desired organism is an essential prerequisite. The enrichment culture technique is a powerful microbiological tool which can be applied for isolation of any specific organism from its natural habitat, provided the selective conditions for its enrichment are known.
A few examples of application of the technique for isolation of different types of microorganisms may be cited:
Enrichment of di-nitrogen-fixing bacteria and endospore-forming bacteria:
If an inorganic salts medium without any nitrogenous compound and containing an organic compound as carbon and energy source is inoculated with a soil or water sample and incubated under aerobic conditions, the selective conditions thus created will encourage the growth of such organisms which can utilize the atmospheric nitrogen gas as the sole source of nitrogen.
Because the atmospheric nitrogen is in molecular form (N2), the organisms enriched are called di-nitrogen-fixers or diazotrophs. The little amount of combined nitrogen that is present in the soil or the water sample used as inoculum might allow growth of non-fixers at the initial stage, but a few passages through the selective media generally eliminate them.
The organisms that can be isolated in this way include different species of Azotobacter, Beijerinckia, Derxia, Azomonas etc. If a similar medium is used and incubated under anaerobic condition, it leads to enrichment of di-nitrogen-fixing anaerobes like Clostridium pastorianum and related species. Enrichment of Clostridia may be further facilitated when the inoculum is pretreated for 5 min at 80°C.
This treatment kills all bacterial vegetative cells, but endospores are spared. On the other hand, a mineral salts-sugar medium with combined nitrogen inoculated with a heat-treated inoculum incubated aerobically is suitable for enrichment of aerobic spore-forming bacteria, mostly different species of Bacillus. By selecting a higher temperature for incubation, it is possible to isolate the thermophilic species of this genus.
The conditions for enrichment of nitrogen fixers and spore-formers are summarised in Table 7.2:
Enrichment of Autotrophic Bacteria:
Autotrophic bacteria include the phototrophic and the chemolithotrophic organisms. The phototrophic prokaryotes include the anaerobic photosynthetic bacteria and the aerobic cyanobacteria. The chemolithotrophic bacteria similarly include a variety of organisms capable of oxidizing different inorganic substrates for obtaining energy.
All autotrophic organisms, including green plants, are able to synthesize organic compounds from CO2. For enrichment of autotrophic bacteria of any type, the media must not contain any organic compound, instead there should be a source of CO2, like bicarbonate.
For enrichment of all phototrophic organisms, the enrichment cultures must be exposed to light. Cyanobacteria are aerobic and many of them can fix atmospheric molecular nitrogen. For their enrichment a mineral salts medium without combined nitrogen, inoculated with a water or soil sample, should be exposed to light under aerobic conditions.
Cyanobacteria carry out oxygenic photosynthesis like green plants. The other group of phototrophic prokaryotes includes the anaerobic organisms which can be divided into two main types — the photolithotrophs and photo-organotrophs. For both, light is the source of energy for CO2-fixation, but whereas the photolithotrophs can use reduced sulfur compounds like H2S as electron-donor, the photo-organotrophs use simple organic compounds like acetate, malate etc. for this purpose.
Conditions for enrichment of different phototrophic prokaryotes are depicted in Table 7.3:
The chemolithotrophic bacteria, like nitrifying, sulfur-oxidising and hydrogen-oxidising bacteria are non-photosynthetic and strictly aerobic. They should be enriched preferably under dark condition to avoid growth of cyanobacteria. The enrichment medium should be composed of inorganic salts including a nitrogenous salt, and an inorganic oxidisable compound acting as energy source.
This compound will depend on the kind of organism to be enriched. For example, for nitrifying bacteria an ammonium salt can be used both as source of nitrogen and of energy. Another group of nitrifying bacteria use nitrite as energy source.
For both types, the enrichment medium is adjusted at an alkaline pH (8.5) and, furthermore, an insoluble acid-neutralizer like CaCO3 or MgCO3 has to be added to counteract the nitrous and nitric acid produced by the bacteria. Carbon dioxide released through interaction of acid and carbonate is helpful for growth. A number of passages through such media are required for adequate enrichment.
Another important group of autotrophic bacteria is the sulfur oxidizers. They can grow under autotrophic conditions using reduced sulfur compounds, like thiosulfate, sulfide or elemental sulfur and produce sulfuric acid. For their enrichment, a mineral salts medium including an inorganic nitrogenous compound and the oxidisable sulfur compound is used. The organisms are aerobic and can tolerate extremely acid pH, although they grow optimally at a neutral pH. Some species are obligately autotrophic and some others can grow also heterotrophically (facultative).
A third group of autotrophic bacteria comprises the hydrogen oxidizing organisms which utilize the oxidation of H2 to water as the energy-giving reaction. They are commonly known as hydrogen bacteria and all of them are only facultatively autotrophic. For their enrichment a mineral salts medium with combined nitrogen and without any organic carbon source contained in a closed vessel is generally employed. The gas phase of the vessel is artificially produced by replacing air with a mixture of H2 (70%), O2 (20%) and CO2 (10%) (v/v).
The conditions for enrichment of the nitrifying, sulfur oxidizing and hydrogen bacteria are shown in Table 7.4:
10. Requirements of Macro- and Micro-Elements for Growth:
These elements must be provided to microorganisms for growth. Water forms the major part of all actively growing living systems and microorganisms are no exceptions. But microorganisms— particularly endospores produced by some of them — can withstand desiccation for a long time without losing viability. Though, in such a state, their metabolic activities and growth are practically absent.
Besides water, carbon is the most important element that constitutes about 50% of the dry weight of bacterial cells. The importance of this element lies in the fact that carbon forms the skeleton of all organic compounds which are present in all biologically significant molecules, like those of proteins, carbohydrates, lipids, nucleic acids etc.
The majority of microorganisms have a heterotrophic mode of nutrition and they derive their, supply of carbon from one or the other organic compounds, like sugars, organic acids, alcohols, complex carbohydrates etc. All such heterotrophs can also fix small amount of CO2 into/several metabolic intermediates (heterotrophic CO2-fixation).
In contrast, the phototrophic and chemolithotrophic microbes draw their carbon requirement fully or for the most part from CO2. Some facultative autotrophs can grow both under lithotrophic (purely inorganic substrates) or under heterotrophic conditions. Some have a mixed type of nutrition, the mixotrophs which can simultaneously utilize CO2 and some organic compound as carbon source.
Next to carbon is nitrogen which forms about 10-15% of the dry weight of microbial cells. Most bacteria can grow only in the presence of combined nitrogen supplied in the medium. A few, including some cyanobacteria, can reduce molecular nitrogen to ammonia and incorporate it into organic acids to produce amino acids.
This property, known as di-nitrogen fixation, is conferred by a special enzyme complex known as nitrogenase. Nitrogen is present in proteins, nucleic acids, cell wall polymers, coenzymes, vitamins etc. Among these, proteins are quantitatively the most important accounting for about 50% of the dry weight of bacterial cells.
Phosphorus is the next major element forming 2-6% of the dry weight of bacterial cells. Microorganisms get their supply of this element from inorganic phosphates provided in the growth medium. Phosphates also help to keep the pH of the medium in a favourable range by a buffering action.
Among the most important cellular constituents that contain phosphorus are the nucleic acids. DNA accounts for 3-4% of the dry weight of bacterial cells and RNA for 10-20%. The energy-rich compounds, like adenosine triphosphate (ATP), guanosine triphosphate (GTP) etc. and phosphorylated sugars are also among important phosphorus containing compounds. Besides, phospho-lipids form the membrane system.
Sulfur is also an essential element for all living organisms. It is found in all proteins as a constituent of the amino acids, cysteine and methionine. Two cysteine molecules can join by oxidation to form a dimer, called cystine (S-5 bond, disulfide bridge).
This reaction is of special significance in imparting a characteristic folding of the polypeptide chain, and also it plays an important role in joining individual polypeptide chains to produce the quaternary structure of protein molecules. Microorganisms get their supply of sulfur from inorganic sulfates present in medium.
Sulfate is reduced intracellularly by an assimilatory reduction pathway to HS – (sulfhydryl) and incorporated into organic compounds. The element sulfur has special significance in case of sulfur-oxidizing chemolithotrophs, like Thiobacillus and the purple and green photosynthetic sulfur bacteria, like Chromatium and Chlorobium.
Some species of Thiobacillus can oxidize elemental sulfur to sulfate and utilize the oxidation energy for chemolithotrophic growth. Purple and green sulfur bacteria utilize sulfide as exogenous electron donor in photosynthesis and in the process they produce elemental sulfur which deposits within or outside their cells.
So far we have considered the role of non-metallic elements in the constitution of microbial cells. Though quantitatively less, metallic elements also form essential parts of microorganisms. Among them, magnesium (Mg ++ ) and potassium (K + ) are required in substantial amounts. Mg ++ is essential for maintenance of the integrity of ribosomes and it acts as a co-factor in many enzyme reactions, particularly those involving ATP.
Moreover, it is present as a part of chlorophyll in all photosynthetic organisms, including cyanobacteria, and purple, green and non-sulfur photosynthetic bacteria containing bacteriochlorophylls. Potassium (K + ) is one of the important intra-cellular elements for maintenance of ionic balance. It also serves as a co-factor in many enzyme reactions and it plays an important role in ribosomal function.
Iron (Fe ++ ) forms an integral part of all haem-proteins, like cytochromes. It is also present in ferredoxin and nitrogenase. Iron bacteria, like Thiobacillus ferrooxidans, are able to grow chemolithotrophically at the expense of the energy of oxidation of Fe ++ —>Fe +++ . Among other metallic elements required by microorganisms are zinc (Zn ++ ), molybdenum (Mo ++ ), manganese (Mn ++ ), calcium (Ca ++ ), cobalt (Co ++ ) and nickel (Ni ++ ).
Zinc and manganese act as cofactors for several enzymes. Molybdenum, together with iron, are integral parts of the enzyme nitrogenase. Another molybdenum enzyme is dimethyl sulfoxide reductase which converts the sulfoxide to dimethyl sulfide. The enzyme has an important role in the sulfur-cycle of nature. Nitrate reductase is still another molybdoenzyme.
Some nitrogen-fixing bacteria possess nitrogenase in which vanadium (Va ++ ) replaces molybdenum. Nickel (Ni ++ ) is required by hydrogen-oxidising bacteria for hydrogenase activity. Some bacteria can synthesize cyanobalamine (Vitamin B12) and they require cobalt (Co ++ ) which is present in the vitamin. Calcium (Ca ++ ) is essential for production of endospores in Gram-positive bacteria. The endospores contain calcium dipicolinate, a compound which is largely responsible for thermo resistance.
Zinc and manganese ions serve as co-factors in several enzyme reactions. Sodium ion (Na + ) is not generally required by most bacteria, although it is sometimes added in culture media mainly for maintenance of a favourable osmotic pressure of the medium.
Most of the common bacteria can tolerate a moderate concentration of NaCl (3-4%), but the marine microorganisms require a higher concentration for growth. There are some extremely salt-tolerant bacteria (the halophiles) like Halobium which need a much higher concentration of NaCl (up to 20%) for maintaining their cell integrity.
Average elemental composition of dry bacterial cells is shown diagrammatically in Fig. 7.12:
11. Physical Factors Influencing Growth:
On the basis of oxygen relation, microorganisms are classified into three major types — aerobic, anaerobic and microaerophilic. While aerobic organisms require oxygen of air for growth, anaerobic organisms — which include mainly bacteria — are unable to utilize oxygen. For some obligate anaerobes oxygen is even toxic. Some organisms which are able to grow both in presence of air or in its absence are called facultative anaerobes. The microaerophilic organisms are also aerobic, but they grow only at a reduced oxygen tension.
The aerobic organisms are capable of oxidizing substrates fully to CO2 and H2O using oxygen as the terminal hydrogen acceptor. In this process of respiration they produce ATP in the electron transport system by oxidative phosphorylation from ADP and inorganic phosphate.
The obligate anaerobes, on the other hand, can oxidize substrates only partially by fermentation, because of the lack of tricarboxylic acid cycle (TCA cycle)-linked electron transport pathway and they produce ATP only by substrate level phosphorylation.
The facultative anaerobes are essentially of two types. Some are capable of changing their metabolism either to fermentation or to aerobic respiration depending on the environmental conditions i.e. whether oxygen is absent or present.
The other group of facultative anaerobes are respiring organisms capable of utilizing oxygen either from air (when air is present), or from oxidized inorganic compounds, like nitrate or sulphate (when air is absent i.e. under anaerobic conditions).
Under anaerobic conditions they carry out the so-called nitrate or sulphate respiration, in which these compounds serve as the terminal electron acceptor in place of oxygen. They produce ATP by oxidative phosphorylation like aerobic organisms.
Microaerophiles are also oxygen-requiring respiring organism, but they can grow only when the oxygen concentration is considerably lower than normal. This is probably due to the oxygen sensitivity of some of their vital enzymes. In an un-agitated liquid culture these bacteria form a layer below the surface where the oxygen concentration is suitable for their growth. In contrast, the aerobic organisms grow at the surface and the facultative anaerobes tend to accumulate at the bottom, if they grow at all.
Bacteria can utilize oxygen dissolved in the growth medium. The solubility of oxygen in water is low and most bacteria are well-adapted to such concentration for normal growth when they grow on agar surface or at the air-liquid interface.
Use of thin layers of culture medium in vessels with a wide surface generally suffices for normal growth of most aerobic organisms. However, when use of larger volumes of medium becomes essential, need for additional arrangement for aeration arises.
For laboratory flask cultures, a mechanical shaker having either a to and fro (reciprocal) or an elliptical movement is commonly used for agitating liquid cultures. When still larger volumes have to be used, arrangements for forced aeration become necessary.
Generally, for this purpose, sterilized air is forced through a sparger producing air-bubbles at the bottom layer of the culture fluid. Larger fermenters are provided with more sophisticated arrangements for aeration. For growing anaerobic organisms, oxygen has to be excluded from the culture medium and the atmosphere existing in the culture vessel. Some anaerobes are somewhat aero-tolerant and they can grow in solid media containing reducing substances like thioglycollate, ascorbic acid or cysteine which presumably minimize the toxic effect of oxygen by interacting with it.
For growing anaerobic organisms in liquid culture, generally closed vessels filled completely with the freshly prepared medium are employed. If an artificial atmosphere (gas-phase) is necessary, it must be oxygen-free. Specially designed containers (anaerobic jars) are available for growing anaerobes.
(ii) Hydrogen-ion concentration:
Hydrogen-ion concentration [H + ] determines the acidity and alkalinity of the culture medium and is commonly expressed as its pH value which is logarithm of the reciprocal of hydrogen ion concentration (gram molecules per liter). The hydrogen ion concentration of pure water is 1/10 7 moles per liter corresponding to a pH value of 7.0 (neutral). As [H + ] increases pH value falls and acidity increases. Decrease of [H + ] from the neutral point results in increase of alkalinity.
The relationships are shown in Fig. 7.13:
Most bacteria prefer a neutral to slightly alkaline medium, while fungi and algae grow better at slightly acidic conditions. For each organism, there is a minimum, an optimum and a maximum pH, permitting growth. The total range may be quite broad extending over 2-3 pH units which corresponds to a difference of 100-1,000-fold in hydrogen-ion concentration. In spite of this wide variation, organisms are able to grow because the cell membrane is hardly permeable to H + or (OH) – . As a result, the cell interior remains more or less neutral.
Microorganisms during growth may produce acidic or alkaline substances in considerable quantities to change the pH of the medium to such an extent which proves inhibitory. For example, the nitrifying bacteria produce nitrous and nitric acids as oxidation products and they turn the medium highly acidic and unsuitable for growth. An opposite situation prevails in case of ureolytic or proteolytic bacteria. They produce ammonia as the end-product which turns the medium strongly alkaline.
For checking these extreme and unfavorable changes in the pH of the culture media, they have to be adequately buffered. Generally, the acidic and alkaline phosphates, like KH2P04 and K2HP04, are used in bacteriological media. An equimolar solution of these two salts produces a pH of 6.8 which is suitable for growth of many bacteria.
If an organism produces an acid in the medium, it reacts with the alkaline phosphate turning it to the acidic form. Opposite is the case when an organism tends to change the medium alkaline by producing a basic substance. Thus, phosphates which are well tolerated by most bacteria are widely used for buffering. At the same time they serve as source of phosphorus.
Though buffering of the medium is generally found to be adequate for checking pH shifts, sometimes, as in the case of nitrifying bacteria which produce profuse amounts of acids, addition of insoluble neutralizers like calcium or magnesium carbonates becomes necessary.
Bacteria, in general, prefer a neutral to slightly alkaline growth medium. But there are exceptions also. The bacteria growing optimally at an acidic pH are called acidophils and those growing preferentially in an alkaline pH are known as alkalophiles.
Not only bacteria (both eubacteria and archaebacteria) but also a number of algae, fungi or even flagellates are endowed with such properties. Many of the acidophilic organisms are simultaneously thermophilic. Some examples of acidophilic bacteria are Acetobacter acidophilum (optimum pH 3), Thiobacillus thiooxidans (pH range for growth 0.9-4.5), Bacillus acidocaldarius (pH range for growth 2-6, opt. 3), Thennoplasma acidophilum (pH 1-2), Sulfolobus acidocaldarius (pH range 1.5-3.5), Non-bacterial organisms which can grow in an acidic pH include Cyanidium caldarium (eukaryotic alga, pH 2-3), Chlorella ellipsoidea (pH 2), Chlamydomonas acidophila (pH 2), Polytomella caeca (pH 1.4 — a flagellate). Some fungi, like species of Cephalosporium, Trichosporon, Aspergillus, Penicillium and Fusarium, can also grow at considerably acidic media.
The true alkalophilic organisms have pH range for growth varying between 8-11 or even more. They include some blue-green algae e.g. Spirullina and Synechococcus, true bacteria, like Bacillus alkalophilus and several other bacilli and some archaebacteria, like Natronobacterium sp., Methanobacterium thermoalcalophilum etc.
Temperature is one of the most important variables for growth. For every organism exists a temperature range which is congenial for its growth. At one extreme of this range is a minimum temperature where growth can start and at the other extreme is the maximum where growth abruptly stops. In between the two extremes, there is an optimum temperature where growth rate is maximum.
On the basis of temperature relations, microorganisms are classically divided into three major groups — psychrophiles, mesophiles, and thermophiles. Psychrophiles are cold-loving organisms growing in marine or alpine habitats. Many of them can multiply at 0°C or even sub-zero temperature and have maximum growth rate between 16°C and 20°C.
Well-known examples of psychrophilic bacteria are marine Photo-bacterium sp. and the iron-oxidizing Gallionella ferruginosa (opt. temp. 6°C). Besides, several marine species of Pseudomonas and some species of Bacillus isolated from glaciers belong to this group.
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