Welcome to the Microbiology Information

Here is a list of culture media used in microbiology laboratory, which is based on the mode of action:

Liquid medium
A liquid culture medium consisting of an aqueous solution of one or more constituents (eg. Buffered Peptone water, Nutrient Broth, Tryptone Soya Broth and so forth).

Solid culture medium
A culture medium containing solidifying or gelling agent (eg. agar-agar) in concentrations varying from 1 to 2 %.

Semi-solid culture medium
A sloppy semi solid medium containing 0.15 % of agar-agar. Commonly employed for motility testing (eg. SIM Agar) or using motility as a selective agent (eg. MSRV).

Culture media by intend of use
The composition of a culture medium formulation determines its purpose.

Preservation medium
A preservation culture medium preserves and maintains the viability of microorganisms over an extended period. During long-term storage the preservation medium protects microorganisms against the adverse influences (eg. Dorset egg medium).

Resuscitation medium
A resuscitation medium is a non selective nutrient rich medium enabling stressed, damaged and injured cells to repair and to recover their full capacity for normal microbial growth (e.g.Tryptic soya agar with 0.3 % yeast extract or Tryptic soy broth).

Enrichment medium
A liquid culture medium provides nutrients for rapid multiplication of microorganisms (eg. Buffered peptone water or Nutrient broth).

Fermentation medium
A liquid culture medium formulated to achieve the nutrients for an optimal yield of specific microorganisms (eg. Yeast) or metabolism product (eg. toxin).

Selective enrichment medium
A selective enrichment medium is formulated to support the multiplication of target microorganism or a group of microorganisms whilst partially or totally inhibiting the growth of accompanying interfering organisms (eg. Muller-Kauffmann Tetrathionate broth with novobiocin for salmonella or L-PALCAM broth for listeria).

Isolation medium
A solid culture medium that supports the growth of microorganisms (eg. Plate Count Agar).

Selective isolation medium
A selective isolation medium which supports the growth of specific target microorganisms, whilst inhibiting other interfering microorganisms (eg. PALCAM agar for selecting listeria or MacConkey agar).

Differential medium
A culture medium which permits the testing of one or more physiological / biochemical characteristics of a microorganisms for their identification (eg. Simmons Citrate Agar).

Identification medium
A culture medium designed to produce a specific identification reaction that does not require any further confirmatory test (e.g.Triple Sugar Iron (TSI) Agar).

General-purpose media
Some culture media may be assigned to several categories. Blood Agar, for example can be used as a resuscitation medium, as isolation medium or as a differential medium for the detection of haemolysis.

Campylobacter is one of those bacteria that causes gastroenteritis, but may also cause systemic illness. It is classified as a pathogen, however it is not seen in the same light as listeria, Salmonella or E.coli. Even the regulators around the world are having doubt if this organism should be regulated.

Who knows, however campylobacter will always be less popular and remain in the shadows of the salmonella, listeria and E.coli.

Symptoms of campylobacter infection include diarrhea with loose bowel movements that maybe bloody, fever and stomach cramps. The most vulnerable to severe infections are the very young, the elderly and malnourished people.

The most frequent cause of the infection is by eating contaminated foods with raw or under cooked poultry being the main culprit. Other sources include household pets, especially puppies and kittens, domestic stock, and contaminated water.

The incubation period for developing the symptoms can vary between 1 - 10 days with this being dependant upon the levels of campylobacter being ingested and the health of the individual. The infected person is infectious for as long as the Campylobacter bacteria are in their faeces, which may be for an average of 2-3 weeks after symptoms are gone. The risk of infecting others decreases when diarrhea is no longer present.

So how do you control this pathogen? Easily, just follow some good personal hygiene.

* Wash hands after handling raw meat, and keep food preparation areas clean
* If possible wear disposable gloves when preparing raw meats.
* Wash hands after gardening, or touching animals and where possible use antiseptic handwash
* Meat, particularly poultry, should be thoroughly cooked
* Do not store uncooked poultry near foods which will be eaten raw, such as salad items
* If pets are sick with diarrhoeal illness, have them treated.

Campylobacter: Molecular And Cellular Biology No Synopsis Available

Here is an interesting product which is developed by the leading microbiology culture media brand OXOID; a listeria result in food using a modified cultural method within 48 hours. Yes that’s right it’s cultural and not one of those ELISA method. I’m not sure what the pricing is like, however if it’s cheaper than an ELISA based method, it’s a winner.

Here’s what they say:

“Oxoid Novel Enrichment Broth – Listeria (ONE Broth) and Oxoid Chromogenic Listeria Agar (OCLA) have been approved by AFNOR (Association Francaise pour la Normalisation) for the detection and differentiation of Listeria monocytogenes and other Listeria species from food samples in just 48 hours.

Together, ONE Broth and OCLA provide presumptive identification of Listeria species in just 48 hours – up to 3 days earlier than traditional methods. The use of just one broth and a single agar plate reduces ‘hands-on’ time, frees up resources and leads to a lower cost per test. ONE Broth and OCLA were validated by AFNOR against ISO 11290-1:1997 and shown to give equivalent results2.”

Source

Bacillus licheniformis is a Gram-positive motile spore-forming rod, facultative anaerobic and belongs to the Bacillus subtilus group of Bacilli. It is an apathogenic soil organism that is mainly associated with plant and plant materials in nature but can be isolated from nearly everywhere in natures such soil, water, food manufacturing plant and so forth. Although its spores are highly heat resistant (100.C for 30 minutes), it is not as resistant as Bacillus stearothermophilus.

Although very very rare, Bacillus licheniformis has been associated in food poisoning in humans with foods such as cooked meat, poultry and vegetable dishes (particularly stews and curries which have been served with rice). Again this is a rare occurrence and not a major concern. Food poisoning by Bacillus licheniformis is characterized by diarrhea, although vomiting occurs in half of reported cases.

Bacillus licheniformis produce proteases and amylases which at high levels can cause the breakdown of short shelf-life foods with starch such as custards, rice puddings, sauces and so forth. Industrially the enzymes produced by Bacillus licheniformis have been extracted for use in household detergents. In the U.S. about 50% of liquid detergents, 25% of powder detergents, and almost all powdered bleach additives now contain enzymes to help break down stains that are otherwise hard to remove with conventional surfactants alone.

Bacillus licheniformis produce also produces penicillinase, pentosanases, bacitracin, proticin, 5′inosinic acid and inosine, citric acid, and substituted Ltryptophan.

Bacillus licheniformis is also a common dairy contaminant being present in raw milk. Monitoring of incoming raw milk for spores is an effective method of determining whether bacillus spores are present in the milk supply. The species has been isolated in pasteurized milk and cream where it can cause bitterness due to the protease enzymes breaking down the milk protein. It has also been reported as a contaminant in UHT milk as well. Although it is very unlikely to survive the UHT sterilization process, it may reside in the environment within the manufacturing plant and therefore Good Manufacturing Practice (GMP) will ensure its prevalence is the environment is reduced. Areas may include dirty valves, seals, heating plates, air vents and so forth.

Bacillus licheniformis also causes ropiness in bread and again monitoring the spore levels in flour may be an effective method of determining whether bacillus spores are present in flour used.

Bacillus licheniformis optimum growth temperature is 30.C; however it will not grow at low pH.

Bacillus No Synopsis Available

When preparing microbiological dehydrated media all aspects of Good Laboratory Practice must be followed. A critical aspect of media preparation is the loss of water or evaporation which must be prevented or minimise. Evaporation does not only change the concentration of the ingredients in the reconstituted medium but vapour coming from the media may contain hazardous / toxic substances which then becomes a occupational health and safety issue.

The following is a guideline on the dissolution or dehydration of any dehydrated culture base medium:

1. Measuring water

It is necessary to measure exact volumes of distilled or deionised or purified water. The measuring cylinders should have accuracy in proportion to the volume to be measured. Eg. 500 mL of water should be measured using a 500 mL or 1 L cylinders but should not be measured in a cylinder of 2 L or greater.

2. Selecting and labelling the flask
The right sized vessel should be 2 to 3 times the volume of the culture medium to be prepared. Volumes of no more than 1 L are preferred. If larger sizes are needed follow the same rule (check first if autoclave fits the needed sized vessel). Overheating of the medium may result when preparing volumes of more than 1 L. Label the vessel (flask) with at least the preparation date, expiry date (helps to identify media immediately that should not be used any more) and identity.

3. Adding small amount water
Approximately a third of the required volume of water is added to a vessel first (this avoids sticking of medium to the bottom and reduces the occurrence of clumping).

4. Transfer of weighed dehydrated medium
The medium should be transferred completely from the weighing boat or clean beaker to the vessel (flask), avoiding airborne dust, and sticking of medium to vessel opening, -walls, and -bottom.

5. Adding remaining water
Progressively add the remaining amount of water and carefully rinse down any material adhering to the walls of the vessel.

6. Check on sticking
All components, except agar-agar and gelatin contained in a dehydrated culture medium, are water soluble. An agar containing medium is dissolved when a transparent agar layer remains on the bottom. A powdered medium sticks quickly to the bottom and components do not completely go into solution even with vigorous shaking. Check before heating the medium - undissolved portions could burn and change the concentrations of the formulation!

Culture media without agar-agar or gelatin can be dissolved usually in cold water, or only require gentle heating. Use should be made of this fact to ensure that the medium is prepared under mild conditions.

7. Soaking agar containing media
Media containing agar should be allowed to soak for several minutes prior to heating (e.g. with mixing).

8. Heating under avoidance of evaporation
Before heating the medium precautions need to be taken against evaporation of water. Vessels (flasks) should be capped e.g. by using non absorbent cotton prop topped with aluminium foil, a loosely tight metal or screw cap. Tightly closed vessels may “explode”, particularly when the reconstitution occurs in a magnetron. It is important that correct glass ware is used.

Check if the medium contains heat labile ingredients. Avoid overheating the media. Nearly all culture media contain peptones or extracts, which are heat sensitive. Overheating of media with a high sugar content and peptones produces Maillard reactions (caramelising) with formation of growth inhibitory substances and darker colours. These media cannot be used, as they were prepared incorrectly.

Heating should be done with frequent agitation to ensure an even heat distribution. Direct contact of a vessel on a heating plate should be avoided as components may get burned before going into solution. Either use a water bath or a cooking pot. Just before a medium begins to boil it should be removed from the heating source. Agar media, particularly those with low agar content, may boil unexpectedly and may flow out of the flask.

Culture media containing agar or gelatin must be heated in order to dissolve completely. Heating should be carried out in a boiling water bath or free-flowing steam (e.g. in a steam pot or a not closed autoclave without excess pressure).

It is common practice to use a heating plate. Direct contact of a vessel on a heating plate should be avoided as components may get burned before going into solution. The medium must be frequently stirred while gently increasing the temperature. Boiling of the medium must be avoided. Overheated media must be discarded.

Although not recommended, medium can be dissolved in a microwave, when the water soluble components, except for agar, are completely dissolved. The microwave heating process should be validated; meaning the optimal time should be assessed for a given type of microwave, a given load, a given type of vessel, and the volume of medium to be prepared. Because a microwave produces high short bursts of heat (a short overheating) it is not considered to be the most ideal way to dissolve a medium. The process is quick and therefore attractive, particularly when non planned small quantities of medium (e.g. Friday late afternoon) have to be prepared. Only the right glassware and caps should be used and vessels not closed too tightly!

9. Check for complete dissolution
Culture media, which are only heated and not autoclaved must be checked for complete dissolution! This is achieved when the viscous solution flows smoothly and if no agar particles are to be seen sticking to the walls of the vessel after shaking. For some culture media a visual turbidity is necessary and wanted (e.g. Bismuth Sulfite Agar). It is essential that the insoluble components should then be distributed as fine as possible to ensure that the turbidity is homogeneous.

10. Cooling
Allow media containing agar or gelatin to cool to 48 ± 2 °C before sterilisation in the autoclave.

Microbiology: An Introduction Media Update No Synopsis Available

HEALTHY adults and children who eat probiotic yoghurts are “wasting their money”, according to an expert in the field.

Dr Elizabeth Furrie, of Dundee University, who is involved in studying the health benefits of probiotics, said there was evidence that they could help people with bowel conditions, but well people did not have to buy the numerous products marketed as a way to stay healthy.

“If you are a normal person and have a healthy gut, there’s absolutely no point,” Dr Furrie told the Society for Applied Microbiology conference in Edinburgh this week. “Don’t buy the stuff in the supermarket … if you are feeling well, don’t waste your money,” she said, adding: “I’ll probably get into trouble now.”

Other experts believe it is worthwhile taking probiotic supplements to ensure levels remain topped up.

Source: News Scotsman

Now here is an interesting microorganism to cause a public recall, Burkholderia cepacia. Burkholderia (previously known as Pseudomonas) cepacia, a nutritionally versatile, gram-negative organism, was first described in 1949 by Walter Burkholder of Cornell University, as the phytopathogen responsible for a bacterial rot of onions. Interestingly, Burkholderia cepacia is now being considered by agricultural microbiologists as an agent to promote crop growth.

The microorganism is inherently resistant to multiple antibiotics, can metabolize diverse substrates, and is found in soil and in moist environments. The organism has a particular predilection for the lung in patients with cystic fibrosis and has emerged as an important opportunistic human pathogen in hospitalized and immunocompromised patients.

Unfortunately, this microorganism was found in certain batches of Comfort Shield Perineal Care Washcloths from Sage Products. The product was distributed to hospitals, medical centers and long-term care facilities in the U.S. and Canada, however there were no known distribution to retail stores.

As a result, Sage Products initiated a recall after receiving and investigating a Canadian complaint on lot 1457 of an off odor. At the present time, Sage Products Inc has received no reports of patient injury. This voluntary recall is being conducted with the knowledge of the Food and Drug Administration.

For more information on the press release click here

There were five fatal cases of listerosis cases in Sydney Australia between 1998 occurring in one particular area North of Sydney. The area is the Hunter which is about 2 hours drive North of Sydney.

Four of the cases had either resided in a nursing home or had periods in hospital with all patient being elderly or immuno compromised.

During an investigative study, Listeria monocytogenes was found in fresh fruit salad at a catering facility in two different batches (opened and unopened) as well as in fruit salad in the processing plant. Environmental swabs were taken in and around the surrounding area and there were some positive results. They included the drains and the food processing trolleys indicating the present of listeria within the environment as well as insufficient or poor hygiene practices to control the pathogen. The strains were then serotype and they matched those from the fruit salad implicated.

Due to the low pH, fruit salad is not a common medium for the growth of listeria and has not been implicated in any outbreak in the past. However unlike other industry such as Dairy where pasteurization is applied to remove pathogen, the processing of fruit salad relies on Good manufacturing Practices (GMP) and hygiene to ensure it’s absent. Poor GMP was evident at both the processing plant and poor hygiene at the catering facility. Rock melon was one of the main ingredients used in the preparation of the fruit salad and it is suspected the source was from dirty rock melons. Rocks melons also have higher pH than other fruits adding to the evidence.

Along with the 5 days shelf-life, any presence of listeria will grow if conditions are favorable. This may occur if the ratio of rock melon is higher as well as poor storage of the fruit salad. Now when you feed this to a high risk group to the equation, you’re asking for trouble.

One question remains unanswered and this was considering the wide geographical area where the fruit salad was delivered why was there not more cases implicated?

Anyhow the investigation received wide publicity and interest from the State Coroner. Interventions following the outbreak have been successful in controlling further cases in the Hunter. They include training and education program and increased regulatory monitoring programs of high risk plants.

Bacillus cereus is a gram positive rod that produces spores and has been recognized as an agent of food poisoning since 1955. There were 52 food poisoning outbreaks between 1972 and 1986 associated with Bacillus cereus were reported, however this is thought to only represent 2% of the total cases which have occurred during that time.

Bacillus cereus causes two types of food poisoning compared to bacterial infections. The first is characterized by nausea and vomiting and abdominal cramps and has an incubation period of 1 to 6 hours. This closely resembles Staphylococcus aureus enterotoxin food poisoning in its symptoms and incubation period and is called emetic toxin and or the “short-incubation”. This is caused by a preformed heat-stable enterotoxin of molecular weight less than 5,000 daltons. The mechanism and site of action of this toxin are unknown. The long-incubation form of illness is mediated by a heat-labile enterotoxin (molecular weight of approximately 50,000 daltons) which activates intestinal adenylate cyclase and causes intestinal fluid secretion.

The short-incubation form is most often associated with fried rice or starchy foods that has been cooked and then held at warm temperatures for several hours. The disease is often associated with Chinese restaurants. In one reported outbreak, macaroni and cheese made from powdered milk turned out to be the source of the bacterium.

The second type of food poisoning results primarily in abdominal cramps and diarrhea with an incubation period of 8 to 16 hours. Diarrhea may be a small volume or profuse and watery. This type is referred to as the “long-incubation” or diarrheal form of the disease, and it resembles more food poisoning caused by Clostridium perfringens. This type of food poisoning is frequently associated with meat or vegetable-containing foods after cooking. The bacterium has been isolated from 50% of dried beans and cereals and from 25% of dried foods such as spices, seasoning mixes and potatoes. One outbreak of the long-incubation form was traced to a “meals-on-wheels” program in which food was held above room temperature for a prolonged period.

The short-incubation or emetic form of the disease is diagnosed by the isolation of Bacillus cereus from the incriminated food. The long-incubation or diarrheal form is diagnosed by isolation of the organism from stool and food as well as the toxin using ELISA based kits such as TECRA.