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Given that bacteria are hypertonic (contain more salts) compared to distilled water, and that they rely on ion concentration differences across the plasma membrane to survive, can I kill a bacterial solution by adding an excessive amount of distilled water to make them explode? If I add distilled water on a surface, are the bacteria on it going to die?
Indeed, bacteria are susceptible to osmotic stress from being in pure water. They are also susceptible to starvation in such an environment.
However, wild bacteria tend to have many mechanisms that protect against this and allow them to survive in spite of being exposed to water. Note, also, that freshwater can serve as a habitat for many species of bacteria: Consider ponds, streams, lakes, etc.
Thus bacteria which you encounter in everyday life will likely have been those that survived osmotic stress in the first place. And drying, and mild disinfectants, and sunlight, and temperature variation… So it is unlikely that water alone will kill them.
Here is a recent article on this topic: http://jgp.rupress.org/content/early/2015/04/07/jgp.201411296.short
And here's one about how osmotic stress affects susceptibility to other antibacterial effects: https://www.sciencedirect.com/science/article/pii/S0740002014001890
Sterile Water vs Distilled Water
Lately, a new question has been popping up online: what’s the difference between sterile water and distilled water?
Sterile water has usually been boiled to kill anything living in the water, but it still has other things in it. Distilled water is made to get you as close to pure H2O as possible – no bacteria, no chemicals, no impurities, etc.
There are some big differences, so we went to our resident water expert and asked him to break it down:
What is the difference between ‘sterile’ water and ‘distilled’ water?
By Eldon C. Muehling
Sterility is a property of water while distilled water is the type of water produced by the distillation process of water treatment. When applied to water, the word sterile means that there are no viable (able to reproduce) bacteria, viruses or any other type of viable microorganism present.
When water is distilled, the first step is to boil the water. The process of boiling kills virtually every microorganism that may be present.
The next step in the distillation process is the formation of steam which is lighter than air. The steam thus rises, leaving any dead organisms behind, and thus producing sterile water, at least for the time being.
If there are any air-borne bacteria in contact with the distilled water some of them will enter the distilled water, but they are not of any great concern. (They can’t live in distilled water and are non-pathogenic but could show up in a standard plate count for bacteria.) Distilled water will also be free of virtually any chemicals.
Learn about all of the amazing uses of distilled water.
Two other processes besides boiling that produce sterile water are chlorination and ozonation. While these processes produce sterile water, the dead bacteria remain in the treated water where they could provide nutrition for any living bacteria that appear. For this reason, excess chlorine or ozone would need to be in the water for the water to remain sterile. Chlorination produces a bad taste and also does not destroy or remove protozoa (single-celled animals). Chlorinated water could, in most cases, contain various chemicals (due to harmful chlorine by-products). Ozonated water does destroy protozoa, leaves no bad tastes, and produces virtually no harmful by-products.
In summary, distilled water is sterile, but sterile water isn’t always distilled. Distillation takes it another step toward perfection.
1. Which of the following is false for the thermal resistance of the bacterial cells?
(a) Cocci are usually more resistant than rods
(b) Cells low in lipid content are harder to kill than other cells
(c) Bacteria that clump considerably or form capsules are difficult to kill
(d) Higher the optimal and maximal temperatures for growth, higher the resistance
Answer: (b) Cells low in lipid content are harder to kill than other cells
2. The most spoilage bacteria grows at ______________.
(a) Acidic pH
(b) Neutral pH
(c) Alkaline pH
(d) All of the above
3. The microbiological examination of coliform bacteria in foods preferably use______________.
(a) Mac Conkey broth
(b) Violet Red Bile agar
(c) Eosine Methylene blue agar
(d) All of the above
Answer: (d) All of the above.
4. Which of the following acids will have a higher bacteriostatic effect at a given pH?
(a) Maleic acid
(b) Citric acid
(c) Acetic acid
(d) Tartaric acid
5. The different ACCs between food categories reflect the ______________.
(a) Potential shelf life
(b) Potential for the microbial growth during storage
(c) The expected level of contamination of the raw material
(d) All of the above
Answer: (d) All of the above.
6. An examination of the presence or absence of organisms refers to ______________.
(a) Incubation of a food suspension in an enrichment medium
(b) Incubation of a food suspension in an enrichment medium followed by inoculation onto a suitable selective medium
(c) Incubation of a food suspension in an enrichment medium followed by inoculation onto a non-selective medium
(d) All of the above
Answer: (b) Incubation of a food suspension in an enrichment medium followed by inoculation onto a suitable selective medium.
7. What are the intrinsic factors for microbial growth?
(c) Oxidation-Reduction Potential
(d) All of the above
Answer: (d) All of the above.
8. Plate count of bacteria in foods generally use the plating medium consisting of ______________.
(a) Peptone, glucose, sodium chloride, agar and distilled water
(b) Yeast extract, glucose, sodium chloride, agar and distilled water
(c) Peptone, yeast extract, glucose, sodium chloride and distilled water
(d) Peptone, yeast extract, glucose, sodium chloride, agar and distilled water
Answer: (c) Peptone, yeast extract, glucose, sodium chloride and distilled water.
9. Enumeration of microorganism refers to ______________.
(a) Non-selective plating, depending on the test.
(b) Either spiral plating, pour plate or spread plate of a food suspension on to a suitable selective agar
(c) Either A or B
(d) None of the above
10. Yeast and mould count determination requires ______________.
(a) Nutrient agar
(b) MacConkey agar
(c) Violet Red Bile agar
(d) Acidified potato glucose agar
Answer: (d) Acidified potato glucose agar.
11. Aerobiccolony count (ACC) is also referred as______________.
(a) Total viable count (TVC)
(b) Aerobic plate count (APC)
(c) Standard plate count (SPC)
(d) All of the above
Answer: (d) All of the above.
12. The time-temperature combination for HTST pasteurization of 71.1C for 15 sec is selected on the basis of ____________.
(b) Coxiella BVurnetii
Answer: (b) Coxiella Burnetii.
13. Suspected colonies of Staphylococcus aureus when grown on Baird-Parker medium shall show____________.
(a) Protease activity
(b) Catalase activity
(c) Coagulase activity
(d) None of the above
Answer: (c) Coagulase activity.
14. NaCl can act ____________.
(a) Transporting nutrients
(b) Antagonist at optimal concentrations
(c) Synergistically if added in excess of optimum level
(d) Both a and b
15. Water activity can act as ____________.
(a) An extrinsic factor
(b) A processing factor
(c) An intrinsic factor determining the likelihood of microbial proliferation
(d) All of the above
Risks of Using Distilled Water
Distilled water lacks even electrolytes like potassium and other minerals your body needs. So you may miss out on a bit of these micronutrients if you drink only the distilled stuff.
Some studies have found a link between drinking water low in calcium and magnesium and tiredness, muscle cramps, weakness, and heart disease. Also, distilled water may not help you stay hydrated as well as other kinds of water.
If you use distilled water for your fish tank, be sure to add a sea minerals supplement to the aquarium. Some coffee fans think that distilled water makes for a purer-tasting cup. But the Specialty Coffee Association of America says that a certain level of minerals is ideal in order to extract the best brew.
Three Main Types of Bacteria
To fully understand this, one needs to know that there are actually three main types of bacteria as to their need for and response to oxygen. They are aerobic, anaerobic and facultative. There are also sub-sets in each of these main types.
Most aerobic bacteria are likely to live where they are exposed to air. Most aerobes require O2 to oxidize sugars and fats in order to obtain energy. Some are facultative and do not. (They can live in air or water.) Micro-aerophiles require minimal O2.
Aero-tolerant organisms are functionally anaerobic, as they don’t have a terminal to accept O2, but can function to some degree in the presence of O2. Most of these are disease causing (pathogenic).
The vast majority of bacteria, perhaps as high as 90%, are non-pathogenic. They are sometimes referred to as the good or friendly type. We need them to be healthy. They are the main type that live in our bodies, mostly in the small intestines where they help us to get energy from food. They are what define our immunity by waging battle against the pathogenic anaerobic types.
Yet, if you look up disease causing bacteria on the internet, you will find at least 41 different categories of disease caused by the bad (unfriendly) bacteria. It is pretty scary when you consider how wide spread they are and how many of them there are in our environment. How would you know which one to test for in your water?
It is not simple, but it is simplified quite a bit, when you consider that there is a surrogate water test for bacteria. That test looks for E Coli in tap water. When there a positive test for Escherichia coli (E coli), that means that the sample is favorable for the presence of other disease causing bacteria. E coli live in the intestines of humans and other warm blooded animals. One strain of E coli 0157:H7 has been linked to severe, acute hemorrhagic diarrhea and left untreated can be fatal.
Ways to Disinfect Water
There are four broad categories of disinfection methods: heat, filtration, irradiation, and chemical methods.
- Boiling water is an excellent method, but obviously, it only helps if you have a heat source. Boiling water can kill some pathogens, but it does not remove heavy metals, nitrate, pesticides, or other chemical contamination.
- Chlorine, iodine, and ozone are most often used for chemical disinfection. Chlorination can leave potentially toxic by-products, plus it doesn't kill all cysts or viruses. Iodination is effective but leaves an unpleasant taste. The use of iodine is not recommended when preparing water for pregnant women or people with thyroid problems. Adding ozone is effective, but not widely available.
- Irradiation is accomplished using an ultraviolet light or exposure to strong sunlight. UV light kills bacteria and viruses but doesn't kill all the algae or cysts of pathogenic organisms. Sunlight is effective if the water is sufficiently clear, the light is bright enough, and the water is exposed to light long enough. There are too many variables to give firm recommendations on the use of this method.
- Microfiltration effectiveness depends on the pore size of the filter. The smaller the pore size, the better the filtration, but it's also slower. This technique removes all pathogens.
Other techniques are becoming more widespread, including electrolysis, nano-alumina filtration, and LED irradiation.
Drinking Distilled and Deionized Water
CC0 Public Domain/pxhere.com
Although some people like to drink distilled water, it's really not the best choice for potable water because it lacks minerals found in spring and tap water that improve the flavor of water and confer health benefits.
While it's okay to drink distilled water, you should not drink deionized water. In addition to not supplying minerals, deionized water is corrosive and can cause damage to tooth enamel and soft tissues. Also, deionization does not remove pathogens, so DI water may not protect against infectious diseases. However, you can drink distilled, deionized water after the water has been exposed to air for a while.
Anti-Bacterial Effect of Garlic (Allium sativum) against Clinical Isolates of Staphylococcus aureus and Escherichia coli from Patients Attending Hawassa Referral Hospital, Ethiopia
Citation: Abiy E, Berhe A. Anti-Bacterial Effect of Garlic (Allium sativum) against Clinical Isolates of Staphylococcus aureus and Escherichia coli from Patients Attending Hawassa Referral Hospital, Ethiopia. J Infec Dis Treat. 2016, 2:2. doi:10.21767/2472-1093.100023
Introduction: Emergence of methicillin drug resistance is evident and global challenge. Seeking for alternative antibiotics which are new, natural, plant based, cost effective and in toxic is the up to date task for global health. Objective: This study was conducted to evaluate the anti-bacterial effect of garlic against clinical and standard isolates of S. aureus and E. coli from patients attending Hawassa University. Methodology: The Antibacterial activity of the crude extract of garlic was investigated against Clinical and Standard isolates of S. aureus and E. coli by an Agar of both dilution and Cork borer techniques. The trial was done in triplicates. Results and conclusions: The results showed that standard S. aureus and E. coli were completely inhibited by 10 mg/ml and 15 mg/ml of agar media respectively and their clinical isolates were completely inhibited by 25 mg/ml, indicating that standard isolates are most sensitive and clinical isolates are least sensitive. Garlic could be used as effective antibacterial agent for these pathogenic microorganisms.
Staphylococcus aureus Escherichia coli Garlic
E. coli: Escherichia coli EPHI: Ethiopian Public Health Institute EHNRI: Ethiopian Health and Nutrition Research Institute Lab: Laboratory MRSA: Methicillin Resistant Staphylococcus aureus MSA: Mannitol Salt Agar Staph: Staphylococcus.
The use of higher plants and preparations from them to treat infections is an age-old practice. Interests in plants with antimicrobial properties has come to use again because of emergence of resistance strains against antimicrobials such as penicillin .
Garlic (Nech- shinkurt in Amharic/Local name/) (Allium sativum L.) is under family Liliacea. It is an erect annual herb with superficial adventitious roots, bulbs composed of a disk like stem . It has long tradition as medicinal plant, started with a direction of preparing a medicinal remedy written in a cuneiform character in about 3000 BC. Scientific investigations of various garlic preparations began in 1939 .
There are a number of studies carried out to assess the value of herbal remedies including garlic preparations for treat of illness [1,2,4].
Recent studies in Ethiopia indicated that garlic has been commonly used in Ethiopian traditional medicine for infectious diseases like tuberculosis, sexually transmitted infections, wounds etc. Besides, its many other culinary applications [1,5].
The antibacterial effect of Garlic (Allium sativum) and other Allium spps has been attributed to S. aureus and E. coli .
This study confirmed that the aqueous extract of Garlic had antibacterial effect against clinical isolates of S. aureus and E. coli.
Garlic as an antibiotic
Garlic is an anti -bacterial agent that can actually inhibits growth of infectious agents and at the same time protect the body from the pathogens. It is known that the most sensitive bacterium to garlic is the deadly Bacillus anthracis which causes the diseases anthrax.
Even the forefather of antibiotic medicine Louis Pasture acknowledged garlic to be an effective antibiotic. Some years later garlic was shown to have similar effect/activity as penicillin. Later studies should similar activity to modern antibiotic including Chloramphenicol. Even the blood of garlic eaters can kill bacteria and it is also reported that the vapor from freshly cut garlic can kill bacteria at a distance of 20 cm! The other, the common and apparently returning diseases tuberculosis was treated with garlic very successfully as invading Mycobacterium tuberculosis is sensitive to several of the sulphur components found in Garlic [6,7].
Allicin is the antibacterial component found in Garlic. A molecular mechanism may be the basis for some of garlic&rsquos therapeutic effects. The researchers were able to study how garlic works at molecular level using allicin, garlic&rsquos main biologically active component . Allicin created when garlic cloves are crushed, protects the plant from soil parasites and fungi and is also responsible for garlic&rsquos pungent smell.
It is a natural weapon against infection that disables dysentery causing amoebiasis by blocking two groups of enzymes, cysteine proteinases and alcohol dehydrogenases. Cysteine proteinases enzymes are the main culprits in infection, providing infectious organisms with the means to damage and invade tissues. Alcohol dehydrogenase enzymes play a major role in these harmful organisms&rsquo survival and metabolism. Because of these groups of enzymes are found in a wide variety of infectious organisms such as bacteria, fungi and viruses. This research provides scientific bases for the notion that allicin is a broad-spectrum antimicrobial, capable of warding off different types of infections.
It is likely that bacteria would develop resistance to allicin because this would require modifying the very enzymes that make their activity possible scientists found that allicin blocks the enzymes by reacting with one of their important components known as self hydyl (SH) groups, or thiols this finding has important implication because of sulfhydryl groups are also crucial component of some enzymes that participate in the synthesis of cholesterol &ldquoGarlic lowers the level of harmful cholesterol&rsquo&rsquo [7-14].
Review on test organism
Staphylococcus aureus is very important pathogen that causes a variety of diseases including skin infections. Gastro intestinal disease (*staph*) food poisoning, toxic shock syndrome and nosocomial infections acquired during hospitalization.
Staphylococcus aureus is isolated on Mannitol salt agar which is a medium that is high in salt (75% NaCl) and contains Mannitol as a source of carbon and energy. S. aureus ferments the mannitol and causes the medium turn yellow.
Escherichia coli is a bacteria that exists naturally as part of normal gut of healthy humans and mammals. It causes enteric diseases. However, there are relatively few strains of this strains pf this organism, which are pathogenic to humans and are associated with food related illness .
Emergence of Methicillin Resistant Strains of S. aureus (MRSA)
There is a growing medical problem due to increasing frequency of infection caused by penicillin resistant Staphylococci. B-lactamase producing strains of S. aureus that are resistant to penicillin first appeared in clinical specimens in early 1950s. Soon thereafter, multiple antibiotic resistance was detected in chemical isolates of S. aureus these strains was resistant to macrolide antibiotic, amino glycoside, and tetracycline.
Plasmids and transposons are clearly important in conferring and transferring antibiotic resistance between bacteria.
Although plasmids and transposons are certainly involved, the actual evolutionary mechanisms underlying this phenomenon have yet to be explained. One this consequence is the emergence of epidemics hospitals strains of S. aureus that is resistant to virtually all useful antibiotics, including methicillin and vancomycin. These strains are currently a significance cause of nosocomial (hospital acquired) infections in parts of the world .
Ethical clearance was obtained from Ethical board of Debub University and support letter were delivered to Ethiopian Public Health Institute and Hawassa Referral Hospital.
Materials and Methods
Garlic solution was obtained not from the whole part of the plant rather on the bulbs. The bulb of Garlic were peeled, weighted and then ligated using pestle while adding small amount of H2O. The extracts then allowed to freeze at -18°C (deep freezing) so as to concentrate the chemical (allicin). Then after freezing, the filtrate was put into lyophilizer till an amorphous powder weighted and then diluted with distilled water and used for the experiment .
Two test organisms (S. aureus and E. coli) which were clinically isolated from patients were collected from Awasssa Referal Hospital.
Two standard pure cultures were collected from EHNRI (Ethiopian Health and Nutrition Research Institute). Both clinical and standard were sub cultured into subsequent Nutrient broth and definite Medias in which they favored i.e., Mannitol salt agar (MSA) for S. aureus and Mackonkey for E. coli were prepared on a slant and on petri dishes .
Cultured of the test organisms were maintained on nutrient both. Briefly, four to six colonies were picked with an inoculating loop and suspended in 5 ml of broth and incubated at 37°C for 24 hours. The turbidity of the broth culture was then equilibrated to match that of 0.5 Macfarlands standards. This provides organisms in the range of 1 × 10 6 to 5 × 10 8 cfu/mol which is pathogenic that used for the test .
Antibacterial activity test
The antibacterial activity test of the crude extract of Garlic against both standard and clinical isolates were carried out by the Agardiffusion method .
Agar diffusion method
The molten agar will mixed with a different concentrations of the test samples at molten state 45-50°C and mixed aseptically with different amounts of garlic extracts to a concentration of 0.25 ml, 0.5 ml, 0.75 ml and 1.5 ml which is equivalent to 5 mg/ml, 10 mg/ml, 15 mg/ml and 25 mg/ml of media. Then, the prepared media were let to solidify. A separate agar plate without sample or drugs was also prepared in order to provide an appropriate growth of organisms. (As the same time as control) Two standard drugs as a positive control were also tested against these microorganisms. These were chloramphenicol 0.30 mg and penicillin 0.30 mg. The negative control used in the cork borer as well as the solvent i.e., distilled water.
Antibacterial effect was determined by direct visual comparison of the growth of the test cultures. All the tests were carried out in triplicate and the results were reported as the averages of these replications.
Cork borker method
In this methods 0.2 ml of garlic extract was mixed with 20 ml of sterile nutrient agar using a mixer (vortex), and then poured into sterile Petri dishes. After congealing, the seeded agar was punched out with a sterile bore (back hole of 10 ml pipette diameter=9 mm) at equally spaced out positions to make four holes. Four of the holes were filled with 0.1 ml of the test sample solution while the fifth with standard antibiotics (chloramphenicol+distilled water) per hole. The plates were then left at room temperature for 2 hours (to favor diffusion over microbial growth) and incubated in an incubator at 37°C for 24 hours. As mentioned before each sample done in triplicate.
The antibacterial activity was evaluated by measuring the diameter of the zone of inhibition using ruler (the media in both methods was prepared according to the instructions that the manufacturer orders (written in the flask that for how many gm of powder how money ml of distilled water is enough).
The results of the susceptibility of the test organisms against the garlic extracts which are stated below in Tables 1a and 1b. Table 2 showed that both clinical isolates of S. aureus and E. coli were sensitive to the concentration of 15 mg/ml (0.75 ml/20 ml of agar media) which is about 80% but about 10% of the organisms were not sensitive for lower concentrations i.e., for 0.25 ml in media.
|Isolates||Micro organisms||Concentration of Garlic/20 ml|
|0.25 ml||0.5 ml||0.75 ml||1.5 ml|
Table 1a: Result of Antibacterial activity of Garlic aqueous extract by agar diffusion method.
|Antibiotic type||Concentration||Size of Clear zone||Organisms presence or absence|
Table 1b: Result of antibacterial activity of Garlic extract compared with standard drugs against test organisms by Agar diffusion+indicates Inhibition-indicates growth.
In addition, larger clear zones were observed at higher concentrations against both microorganisms. Comparatively, Garlic extracts inhibited bacterial growths than Tetracycline and Penicillin.
The result showed the both clinical and standard isolates of S. aureus and E. coli were highly sensitive to concentrations of 0.75 ml/20 ml of agar media in using diffusion method and Cork borers. Moreover, unlike clinical isolates of S. aureus, clinical isolate of E. coli was a bit resistant/not sensitive/ at concentration of 0.5 ml/28 ml of media. This could be in regard with the nature permeability of E. coli, which means 20% of membrane of E. coli is made of lipid while that of S. aureus is only made of 2% lipid . Therefore, the garlic extract was more important for the prevention of resistant S. aureus which is currently becoming a challenge developing resistance to many commercially available drugs like penicillin.
In this study we have observed that, as the concentration of the garlic extract increases we have seen efficiency increased and hence inhibition and growth of test bacteria has been diminished. As observed from the above tables, Larger clear zones at higher concentrations and lower clear zones at lower concentrations. This implies that, Garlic has both bacteriostatic and bactericidal effect.
Based on the results of this study which showed garlic to make large clear zones than currently available antibiotics used in the study Garlic could be used as an effective antibacterial agent in Ethiopia where S. aureus is known to be resistant.
It could be made as a tablet in the best concentrations and affordable dosages so that it can be used as medicine to these two pathogenic gastro intestinal enteric. In the era of those drug resistant bacteria, we need to focus on alternative drugs that have long history to avoid such emerging diseases and that could be easily available and affordable.
One can use garlic as a member of the daily diet for better health especially the fresh garlic. This research can be used as a base to well identify the actual allicin minimum inhibition concentration.
Consent to Publish
The authors declare that they have no any competing interests.
EA and AB designed the study methodologies and EA carried out the investigation and AB edited the manuscript. All authors approved the manuscript.
Availability of Data and Materials
Materials used for the study were stored in Hawassa University, Department of Biology Laboratory. But the files are fully presented in this manuscript.
First of all, I would like to thank my God who gave me the strength and patience to do the research. Then, I would like to appreciate the staff member of Ethiopian Health and Nutrition Research Institute/EHNRI/ especially the Drug Research Laboratory Heads Dr. Asfaw Debela, Dr. Dawit Dikasso and W/O Hirut Lemma for their valuable inputs to carry out the research at EHNRI. Next, I would like to thank my advisor Ato Asefaw Berhe who facilitated ways to work on this project and shared me important ideas for the research. Last but not least, I like to thank staff members of Hawassa Referral hospital Lab department and Laboratory Department of EHNRI for their assistance while I was preparing cultures and collection of standard and clinical isolates of my study microorganisms.
Prions and endotoxins: reprocessing strategies for reusable medical devices
10.8.1 Endotoxin removal strategies and sterilization processes
Endotoxin is not reliably destroyed by disinfection, steam sterilization processes, or ethylene oxide sterilization. And although recent research on the use of plasma exposure to inactivate endotoxin shows promise for the future ( Shintani et al., 2007 Hasiwa et al., 2008 ), currently available low-temperature plasma technology sterilizers may not have the same source gas for plasma generation as that used in research (i.e. nitrogen vs hydrogen peroxide) and may not be validated for endotoxin inactivation. Therefore, when protocols for preparing water for injection or for instrument reprocessing call for sterilization, the general and most practical strategy is to prevent endotoxin contamination of items to be sterilized rather than try to remove it. Endotoxin contamination sources include water used as a solvent, water used in instrument cleaning and terminal reprocessing, packaging components and raw materials or equipment used in production ( FDA, 1985 ). As an example, water that contains high numbers of gram-negative bacteria will be expected to have a high concentration of endotoxin, and if such water is used during instrument reprocessing it follows that this endotoxin will be deposited onto the surfaces of the instruments. Steam sterilization is not an effective depyrogenating process, so endotoxin as a clinically important biocontaminant remains active on the surgical instruments. Therefore, the logical endotoxin control strategy for heat-stable instrument sterilization is to control the bacterial contamination levels in the water used to rinse the cleaned instruments. Water containing ≤ 100 EU/mL has been determined to leave very little endotoxin residue on instrument surfaces, thereby minimizing the potential for a pyrogenic reaction in the patient after surgery ( AAMI, 2007 ). Therefore, central sterile departments in hospitals and other healthcare venues will have a water treatment system in place to provide water that meets the quality requirements for sterile instrument reprocessing.
Water treatment systems typically consist of three components: (1) a pretreatment stage (2) a water treatment process and (3) a distribution system. Pretreatment is used to remove hard contaminants such as sand and other insoluble objects (e.g. bits of rock). Incoming water is treated to remove organic and soluble inorganic impurities, including the antimicrobial chemicals used by the municipal water authority to treat the water for community use (e.g. chlorine, monochloramine). There are three water treatment options available for use: (1) deionization (2) reverse osmosis (RO) and (3) distillation. Depending on local conditions of the municipal water, it may be necessary to validate the performance of the distillation unit, as high levels of organic contaminants such as endotoxin can diminish the unit’s effectiveness. Similarly, it may be necessary to have several RO filtering units connected in series in order to provide effective microorganism and endotoxin removal, when municipal water has a high heterotropic plate count (HPC) reading ( FDA, 1985 ). RO filtering units should be disinfected regularly to prevent bacterial build-up. The treated water is then distributed to the various points of use within the central sterile department via a dedicated distribution system. One important quality control task is to prevent any amplification of bacteria and establishment of biofilm in the distribution system. The presence of large populations of planktonic gramnegative bacteria can eventually lead to increase in endotoxin concentration downstream from the main water treatment. Two methods used to keep the distribution system clean are the disinfection of the pipes on a periodic basis and the continual recirculation of the water. Disinfection of the interior pipe surfaces can be accomplished through the use of ultraviolet light (UV), ozone, hot water temperatures, or disinfectant chemicals such as hydrogen peroxide (H2O2) or peracetic acid ( AAMI, 2007 ). Periodic microbiological monitoring is an important part of the effort to maintain water quality in the distribution system, as several factors can enable any residual bacteria to increase in number (e.g. increase in water temperature, distribution system bacterial build-up, use of holding tanks). Should an increase in bacterial counts occur (detected via the use of (HPC) obtained by conventional water sampling methods), the problem can be identified quickly and remedial action to lower the bacterial count can be initiated ( APHA et al., 1998 ).
Endotoxin is removed during the water treatment process. Of the available treatment methods, RO and distillation are each more effective for endo-toxin removal compared with deionization, as deionization does not remove microorganisms or organic matter ( AAMI, 2007 ). The finished water from a central sterile department water treatment process is described as a high-purity water (AAMI, 2007), and it is typically indicated for the final rinsing of cleaned critical and semi-critical medical and surgical instruments. According to the Association for the Advancement of Medical Instrumentation (AAMI), instruments rinsed with high-purity water are expected to have < 20 EU residual on their surfaces ( AAMI, 2007 ). Additionally, in order to keep residual bacteria counts and endotoxin concentrations to a minimum, high-purity water is generated on-demand. In some specific instances (i.e. the rinsing of delicate ophthalmic surgical instruments), sterile distilled water is recommended for the final rinse ( ASCRS and ASORN, 2007 ).
High-purity water is occasionally checked for endotoxin levels, with acceptable concentrations being those < 10 EU/mL ( AAMI, 2007 ). The bacterial endotoxins test (BET) is an assay method for active endotoxin in which a liquid sample is mixed with Limulus amebocyte lysate (LAL) reagent the resulting proportional reaction is measured via visual, turbidimetric, chro-mogenic, or other validated means of detection ( AAMI, 2010a ). The gel-clot technique (a visual method) is simple to perform, requires minimal equipment and data analyses are easy. Details for this and other test methods are beyond the scope of this chapter, but these are summarized in the ANSI/ AAMI standard ( AAMI, 2010a ) and in Chapter 85 in the US Pharmacopeia standard (US Pharmacopeia, 2011a ). Water samples are collected from the following locations within the water treatment system: (1) the reprocessing (cleaning and rinsing) area (2) storage tank (if this equipment is present) and (3) immediately downstream from the treatment equipment (e.g. the RO filtering unit). Endotoxin levels are typically checked when the water treatment system is installed and whenever any modifications or repairs are made. If elevated endotoxin levels are detected, remediation is initiated and the system is tested repeatedly until the levels fall below the action level of 10 EU/mL.
Many of the instrument reprocessing procedures are either automated or involve use of equipment with some manual activity. Although high-purity water is not indicated for the initial instrument cleaning processes, it is nevertheless important to keep the cleaning equipment fully maintained so that all surfaces are kept clean and any fluid reservoirs (e.g. ultrasonic baths) are drained and replaced regularly or when it is evident that the solution has a high organic matter load. These steps will help to keep residual waterborne bacteria levels to a minimum.
The quality of the water used to generate steam is important for the success of the steam sterilization process. Water for steam must be treated to remove minerals, suspended solids and other contaminants to ensure production of as close to 100% saturated steam as is possible ( AAMI, 2010b ). However, it is not necessary to use high-purity water for steam generation. Studies have shown that despite the presence of low numbers of microorganisms in water intended for steam production, instruments exposed to steam from such water do not appear to have significant levels of residual endo-toxin ( Martin and Daley, 2001 Steeves and Steeves, 2006 ). Consequently, monitoring the water intended for steam production for bacterial counts and endotoxin levels is generally not recommended ( Whitley and Hitchins, 2002 Flocard et al.,2005 ).
What is a bacterium?
Most people have heard of bacteria but know very little about them. In general, bacteria are considered by many people to be dangerous or harmful organisms which should be killed. However, this is a misconception as most bacteria are either beneficial or neutral with respect to our lives and the planet on which we all live.
Figure 1: A photomicrograph taken with a light microscope showing a human hair (the long dark object) and many bacteria which are much smaller than the hair. Some of the bacteria are shown at the tips of the blue arrows.
Where bacteria live
Bacteria are extremely small organisms. You cannot see them without a microscope. Figure 1 shows a human hair and bacteria as seen in the light microscope. Although they are very small, bacteria can do some interesting things. Many bacteria can swim using a propeller-like structure called a flagellum. Some bacteria can hold onto surfaces by using projections from their surface called pili. These structures are shown in the illustration in Figure 2.
Many bacteria live in environments in which they need to be resistant to changes in their surroundings. Most bacteria are surrounded by a cell wall which gives them strength so that they are not killed by detergents or distilled water (as found in rainwater) or other stressful things.
Many bacteria live in soil. Some of these bacteria help plants to get the nutrients they need by converting the nitrogen gas in the air into ammonia which the plants can use. Other bacteria are involved in the mineral cycles which keep the ecosystem running. They help to cycle carbon, iron, sulfur, phosphate, and nitrogen. Some bacteria are photosynthetic although they may not be the same colour as photosynthetic plants they are often pink or purple as well as green.
Bacteria can also make things we use such as Swiss cheese and yogurt, while other bacteria can be used in industrial processes to make vitamins and medically useful products such as insulin.
Some can live in extreme environments such as under the Antarctic ice sheet or in hot springs at Yellowstone National Park. The bacteria under the ice sheet are eaten by the small crustaceans called krill which are in turn eaten by whales and other sea animals. This means that the whales are indirectly dependent on the bacteria for their food and thus their lives.
There are bacteria that can use things which we find unusual as food, for example, getting their energy from oil. These bacteria are quite destructive when they get into oil storage tanks. However, they are very useful when there has been an oil spill on a beach as they can be spread on the spill and will convert the oil into compounds which are less harmful to the environment.
Figure 2: A diagram showing the some of the main features of a bacterial cell Figure 3: A picture of a staphylococcal boil on a human hand. The area around the boil is swollen and quite painful to touch.
Bacteria and the human body
We provide the environment in which many species of bacteria live. Some bacteria live on our skin. Remember that bacteria are exceedingly small so different parts of our skin provide vastly different environments for bacteria. Many bacteria live in our digestive tract and provide helpful functions in the digestion of food and the production of vitamins such as vitamin K. If you have taken oral antibiotics, you know that they often give you a bit of an upset stomach. This is because they killed many of the useful bacteria in your intestines. Doctors sometimes suggest that after you take an antibiotic you should eat food containing a probiotic or eat something which contains useful bacteria such as yogurt as these bacteria may then replace the bacteria which died.
Bacteria and disease
However, as you know not all bacteria which grow in or on the human body are helpful. Some of them cause diseases such as diarrhoea, sore throats, boils on the skin (see figure 3) and various other diseases. These bacteria live using nutrients supplied by your body. They may kill some of your cells to get the nutrients they contain. Your skin, stomach acid, and mucous in your nose and throat act as barriers to prevent bacteria from getting inside you. When they do get in, your white blood cells try to eat the bacteria and kill them. In turn, the bacteria try to kill your white blood cells. The white pus and the white material at the centre of a boil like the one seen in Figure 3 contain bacteria and live and dead white blood cells.
Antibiotics and bacterial disease
Antibiotics are chemicals which specifically stop the growth of, or kill, bacteria. One of the first antibiotics was penicillin. This chemical prevents the bacterium from making new cell wall. If the bacteria try to grow and get larger but cannot make new cell wall, then they end up with a gap in the wall. This gap makes the bacteria fragile, and they burst and die. Since we humans do not have cell walls surrounding our cells, we are unaffected by penicillin. Thus, we can safely take this drug when we have a bacterial infection. (It should be noted that viruses do not have cell walls and so penicillin does not kill them). Other antibiotics act in a similar way to block something that bacteria need that humans do not have or do not need.
Bacteria are very small organisms with a simple structure. They can be useful or harmful depending on the kind of bacterium and on the situation in which they are found. Antibiotics can be used to kill bacteria which cause disease without harming people because antibiotics block the production (or activity) of structures which are found in bacteria but not in humans.
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