Welcome to the Microbiology Information

Legionella are bacteria that are common in the environment such as rivers and lakes as well as artificial water systems which includes hot and cold water systems such as storage tanks, pipework, taps and showers. In the environment the occurrence of legionella in groundwater is also common.

With Legionella we usually associate it with larger water systems such as those in factories, hotels, hospitals and museums, and cooling towers. However, they can also live in smaller water supply systems used in homes and other residential accommodation.

In addition, other potential sources of legionella include spa and whirlpool baths, humidifiers in factories and fire-fighting systems such as sprinklers and hose reels. Legionella can survive in
low temperatures, but thrive at temperatures between 2OoC and 45oC. At high temperatures, greater than 6O0C legionella will be killed.

Legionella causes Legionnaires’ disease and it is a potentially fatal form of pneumonia. It can affect anybody, however the risk is higher for those over 45 years of age, smokers and heavy drinkers, those suffering from chronic respiratory or kidney disease, and people whose immune system is low or impaired.

Legionellosis is the collective name given to the pneumonia-like illnesses caused by legionella bacteria, including the most serious and well-known Legionnaires’ disease, and also the similar but less serious conditions of Pontiac fever and Lochgoilhead fever.

If you suspect an area may have the potential for Legionella, then it is best to get the water tested using an accredited water testing laboratory.

The 3M Petrifilm Environmental Listeria plates sounds like a great product. It is a sample-ready culture medium containing selective agents, nutrients, a cold-water-soluble gelling agent, and a chromogenic indicator that facilitates Listeria colony detection. 3M claims the plates were designed to analyze environmental samples and to help increase the efficiency of monitoring plant sanitation. The presence of indicator Listeria such as Listeria innocua provides evidence that environmental conditions are suitable for the occurrence of Listeria monocytogenes. As the two usually co-exist together.

Here is the interesting remark, according to 3M the Petrifilm Environmental Listeria plate detects the majority of environmental Listeria, consisting of Listeria monocytogenes, Listeria innocua, and Listeria welshimeri.*. Here is the sub-clause about the other strains of listeria. “* For further information on the prevalence of Listeria species, please contact the official 3M Microbiology representative nearest you. L. ivanovii, L. grayi/murrayiand L. seeligeri grow but do not form typical colonies.”

That’s right, although all listeria colonies will grow, not all strains will form typical colonies and therefore as a selective medium this is deceptive marketing. Interestingly, the first interpretation guides did not mention the strains in question. That is Listeria ivanovii, Listeria grayi/murrayi and Listeria seeligeri.

Normally a good microbiologist will pick this up, however there are some managers or technical personnel who do not have a clue about this product and will be persuaded by one of the 3M sales representative.

Although Listeria monocytogenes is the main organisms of interest, some regulatory bodies will want all listeria species to be detected in their product. Therefore if this is the case then the 3M Petrfilm Environmental Listeria Plates is not a good option for environmental monitoring and I would stick to the normal ELISA based methods. If on the otherhand, you are not concern with other species of listeria, then this product is a great product.

My point is 3M would be better to develop two different plates:
1. a plate that detects Listeria monocytogenes / innocua only
2. a plate that detects all listeria strains (including Listeria ivanovii, Listeria grayi/murrayi and Listeria seeligeri.)

Finally any pathogen testing must be conducted by qualified personnel’s in an accredited laboratory otherwise the risk of further contamination is raised.

Would I buy this product?
No as some listeria strains do not form typical colonies.

Listeria: A Practical Approach to the Organism and Its Control in Foods No Synopsis Available

Yersinia enterocolitica is a gram -ve bacterium that belongs to a family of rod-shaped bacteria. Other species of bacteria in this family include Yersinia pseudotuberculosis, which causes an illness similar to Yersinia enterocolitica and Yersinia pestis, which causes plague.

Yersinia enterocolitica can cause illness in humans, however only a few strains are implicated. These strains are usually found in animals with the majority in pigs. Other strains are also found in many other animals with lower frequency and they include rodents, rabbits, sheep, cattle, horses, dogs, and cats. In pigs, the bacteria are most likely to be found on the tonsils.

Yersinia enterocolitica is commonly present in foods but with the exception of pork, most isolates do not cause disease. Similarly to Listeria this organism is psychrotrophic meaning it can grow at refrigeration temperatures. Although rare, contamination in pasteurized milk has been documented and I’m surprised that it has not been included as an organism of interest in dairy companies around the world. The organism is sensitive to heat (5%) and acidity (pH 4.6), and will normally be inactivated by environmental conditions that will kill coliforms.

The illness caused by Yersinia entercolitica is called Yersiniosis and is common in children. Symptoms are similar to salmonella infection and include fever, abdominal pain, and diarrhea, which is often bloody. The symptoms appear 3 to 7 days after ingestion of the implicated food or exposure and may last up to 3 - 4 weeks.

In older children and adults, abdominal pain may occur predominantly on the right hand side and may be confused with appendicitis. In rare occurrences and in small proportion of cases, complications such as skin rash, joint pains or bacteria infection of the bloodstream can occur (sepecemia).

If you have symptons of salmonella food poisoing then you should consult with your doctors and get treatment. Symptoms for adults include signs of dehydration, such as thirst and decreased urination, lethargy, dry mouth, feeling faint on standing. In addition there may also be fever, severe abdominal pain and /or bloody diarrhoea.

Recovery from Salmonella infection usually occurs within a few days and without antibiotic treatment, however infants, the elderly and people with immune suppression such as those who recently had surgery may require antibiotics.

Mild gastroenteritis is a common illness, which can be particularly serious in young children. Though antibiotic treatment is seldom recommended, there are several treatments that are useful for gastroenteritis of any cause. The following are general recommendations for the treatment of gastroenteritis:

Ensure they receive plenty of fluids. Oral rehydration solution is highly recommended. It is available at pharmacies and should be administered following the instructions on the packaging.

For mildly unwell children, diluted juice or carbonated (fizzy) drinks (one part to three parts water) or cordial (even more diluted) can be given.It is most important that the drinks given to the mildly unwell children are diluted properly.

If symptoms persist then a doctor should be consulted. Please note, medicines used to prevent vomiting or diarrhoea should not be given (especially in children) except where specifically advised by a doctor. Breast-fed babies should continue to be breast fed with extra fluids between feeds. Finally, children on formula or solid diets should not have food withheld for more than 24 hours.

Water is the main ingredient used in the preparation of microbiological media. Therefore water used must be purified and/or deionized water free from any nutritive and/or toxic substances so that there is no inhibition of the traget micro-organims. Purified water shall have a resistivity of at least 300 000 Wcm and the conductivity should be less than 10 mS (microSiemens).

Tap or potable water must not be used. In some areas tap water may be contaminated and may contain relatively high amounts of heavy metals and /or chlorine. Even in very low levels, these can cause precipitation problems and may inhibit the growth of microorganisms.

If the distilled water is prepared from chlorinated water, it is necessary to neutralize the chlorine prior to distillation. This is achieved by adding sodium thiosulphate.

The distilled water can be stored in containers. These should be produced from inert materials (e.g. neutral glass, polyethylene etc.). The containers must be free of any inhibitory substances prior to their initial use. If, during storage, no precautions are taken, atmospheric CO2 will dissolve, making the water acidic. Also algae may grow quickly in water tanks and their metabolites can inhibit growth of microorganisms.

In some cases it may be necessary to use freshly prepared water, free of dissolved carbon dioxide.

Water processed through an ion exchanger (de-ionized), may have high microorganism content. De-ionized water should not be used without verifying that it does not contain microor-ganisms. Filtering the water is not enough as water may contain substances inhibitory to the growth of particularly fastidious microorganisms.

In Standard Methods for the Examination of Water and Wastewater a test for the bacteriological suitability of laboratory water is described.

If your microbiology laboratory is accrediated to the ISO 17025 standard, then there are a few requirements you must fullfill when you receive your culture media.

Firstly, upon arrival of your shipment of dehydrated culture media and/or supplement, the laboratory staff must check the following:
1. The name of the medium,
2. The lot#, and the expiration date.
3. The product documentation accompanying the shipmen i.e. Certificate of Analysis (CofA), BSE/TSE Certificate (country of origin of the material), Material Safety Data Sheet (MSDS), the CE label (for Europe: if a medium is used for clinical testing), and the technical data sheet.

A comparison of documentation, in particular certificates from different manufacturers/suppliers help in selecting a brand supplier.

The laboratory staff signs off on the receipt check and documents the following information, for example, in an inventory log book and also on the label of the product: the date of receipt and a code that product passed the receipt check.

Finally, the results of the monitoring and seal check is documented in the inventory log book.

Did you know that among the E. coli human pathogens, Verotoxin (Shiga-like toxin) forming strains (VTEC) have gained in importance in recent years. The group of enterohaemorrhagic E. coli (EHEC) with its highly pathogenic serovars 0157:H7, 026, 0103, 0111, 0145, and other strains are of particular concern.

Verotoxins can be classified into two main categories Verotoxin 1 (VT1, SLT1, Stx1) and Verotoxin 2 (VT2, SLT2, Stx2). EHEC strains may produce either VT1 or VT2 only or both VT1 and VT2 simultaneously. EHEC are capable of initiating life threatening illnesses, particularly in those with immune deficiency, young children and the elderly.

Detection of verotoxin is the easist way in which you can determine if the E.coli of interest is a pathogen of real concern.

E.coli is common everywhere with the main sources of infection being contaminated, raw or insufficiently heated foods of animal origin, e.g. meat and dairy products.

The reservoir for EHEC is the feces of cattle, sheep and goats. These microorganisms can enter food during the processing of meat and dairy products if hygienic conditions are inadequate. The drastic increase in the incidence of food infection caused by E. coli 0157 demands reliable and rapid methods of detection. In addition to traditional culture methods, immunological techniques are becoming more useful due to their improved specificity and sensitivity.

Duopath® Verotoxins is an immunological screening test based on the immune flow principle. The Duopath® Verotoxins GLISA test is an immunochromatographic rapid test intended to be used in food-analysing laboratories for the qualitative detection of Verotoxins (Shiga-like toxins) 1 and 2 from Verotoxinogenic E. coli (including E. coli O157:H7) isolated from food enrichments using FDA, USDA or other food enrichment methods.

This test has been validated and received AOAC approval for detection of Verotoxins 1 and 2 from isolated Verotoxin-producing E.coli (including E. coli O157:H7). Duopath® Verotoxin is also intended to be used in clinical laboratories for the qualitative identification of Verotoxins 1 and 2 (Shiga-like toxins 1 and 2) produced by E. coli isolated in cultures derived from clinical stool specimens. The identification aids in the diagnosis of diseases caused by enterohemorrhagic E. coli infections.

Did you know that the composition and packaging of the medium determine its susceptibility to deterioration during storage.

For example, the presence of blood, antibiotic or heat sensitive inhibitors will severely limits the useful life of a medium. The optimum storage temperature for the majority of prepared media is between 4°C to 6°C. When temperatures exceed these ranges, the shelf-life of the medium decreases

Most liquid media do not deteriorate at 4°C for many months but some have a tendency to form deposits, especially those of double strength. Some liquid media have short shelf life even at 4°C, for example, tetrathionate broth.

A volume check should be made on older stock that may be susceptible to evaporation.

It is important to prevent exposure of culture media to sunlight, as this may adversely affect their performance through the formation of peroxides, or by affecting the stability of dyes. Most solid media keep for many months if stored in an airtight container, however there are notable exceptions (e.g. some formulations of Baird-Parker medium).

Agar gel is normally very stable but in media with a pH of less than 5 softening may take place during sterilization, subsequent storage, or re-melting. Therefore storage of agar plates presents two main problems: 1. Contamination and 2. Dehydration.

The length of time that plates can be kept before use depends on the ability to prevent contamination and to minimise loss of moisture. Both of these effects may be reduced by wrapping and sealing plates in plastic bags or cellophane during storage at 4°C, and by implementing effective stock rotation programs within the laboratory.

http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Media_Prep/010_recap_securely_P7120130.JPG

Supplement: Blackboard Subscription Access Card - Microbiology: An Introduction Media Update 7/e No Synopsis Available

The Gram Stain

Gram staining is a procedure that microbiologists are taught in their first practical classes. It is the core of microbiology and fundamental to bacterial classification and identification.

The Gram-positive cell retains the crystal violet - iodine complex made in the first steps of the procedure, despite the subsequent washing, decolourising and counterstaining. Gram-negative cells lose the complex and take on the colour of the counterstain.

Gram reactions can sometimes be misleading, giving either a false positive or a false negative result. There are usually three reasons for a false reaction. The culture is not pure, the age of the culture is old or there is a problem with the method applied.

1. Impure Micro-organism

If Gram-positive and Gram-negative cells appear on the same slide, the first step should be to check the purity of the culture. This can be performed by visually looking at the different colony types. Mixed cultures can cause this reaction, however there are some which are variable in their gram reaction.

2. Age of the Culture

Some Gram-positive bacteria appear Gram-negative when they have reached a certain age. This can vary from hours to days.

On the other hand some Gram-negative bacteria become Gram-positive when the culture is quite old. If you suspect this is the case, stain at 2 or 3 different ages and see when the change occurs. Gram reactions should be determined on very young culture, after growth on the plate has become just visible. Some micro-organisms are truly Gram-variable, appearing Gram-positive or Gram-negative according to the conditions.

3. Problems with the Gram Staining Method

If you suspect a problem with the method, check it against a reputable source or text. Here are some hints on performing a correct Graim stain:

ï‚· Greasy slides lead to poor staining, as water solutions run into droplets. Water spreads out in a thin uniform film on slides that are grease free. New slides are generally not clean enough for staining. Slides should be cleaned in alkaline potassium permanganate and then washed with distilled water or 70% alcohol. Avoid touching the slide with fingers by using forceps when handling.

ï‚· When preparing the smear, avoid overcrowding of cells as this prevents proper decolourisation during the washing steps. Cells should lie separately, with approximately 100 cells per microscopic field. A good smear should be not more than barely visible on the slide after staining.

ï‚· Heat fixing smears can sometimes cause Gram-positive cells to stain negatively. If this is the case try methanol fixation; air dry the fresh culture onto the slide, cover with methanol and allow to evaporate at room temperature, then proceed with staining. Gram-positive bacteria fixed in this way are more resistant to decolourisation.

ï‚· Stock solutions of I2 - KI in water are unstable. Store below 25ï‚°C away from light for not longer than 3 weeks. If 12 degrades, Gram-positive bacteria will stain negative. Use freshly prepared solutions where possible or add polyvinylpyrolidine at 1%; this complexes with the 12 and makes it quite stable.

 When washing the slide, don’t run water directly onto the smear. Dip the slide into tap water in a 250mL beaker and have tap water running into the beaker constantly.

 Examine preparations with the oil immersion objective of the bright field microscope, with the condenser fully open. Don’t use phase contrast, as this does not allow recognition of true colours.

ï‚· Weakly Gram-positive bacteria are best detected if the preparation is not counterstained. In this case phase contrast or bright field can be used to differentiate cells.

Gram Stain - A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References This is a 3-in-1 reference book. It gives a complete medical dictionary covering hundreds of terms and expressions relating to gram stain. It also gives extensive lists of bibliographic citations. Finally, it provides information to users on how to update their knowledge using various Internet resources. The book is designed for physicians, medical students preparing for Board examinations, medical researchers, and patients who want to become familiar with research dedicated to gram stain.If your time is valuable, this book is for you. First, you will not waste time searching the Internet while missing a lot of relevant information. Second, the book also saves you time indexing and defining entries. Finally, you will not waste time and money printing hundreds of web pages.

Did you know E. coli are divided into four different subgroups. These are A, B1, B2 and D based on a
range of factors including genetics, phenotype and ecology. Group A and B1 strains occur in all vertebrate hosts and in water; Group B2 strains are found in warm-blooded vertebrates with hindgut fermentation; Group D strains are found in warm-blooded vertebrates.

In general, A and B1 strains appear to be generalists, acquired by their hosts from the environment, and which colonise
well but persist poorly. B2 and D strains, which encode the most virulence factors, appear to be acquired from other host animals, colonise poorly but persist well.

The distribution of the subgroups in human populations varies in different parts of the world. The prevalence of subgroups in humans may change with age in population and there appears to be a gender effect.

In water, B1 strains are by far the most dominant, with B2 and D strains rarely found. Interestingly, B1 strains have similar sugar utilisation patterns and optimal growth temperatures whether isolated from water or faeces, while A strains differ in both these characteristics depending on their origin. B1 strains appear to survive the transition to the aquatic environment best, with B2 and D strains surviving relatively poorly in water. Exposure to the aquatic environment appears to select for a subset of group A strains.