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.

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

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

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

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.

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.

Manufacturing of peptone is initiated when protein is broken down in a digestion or hydrolysis process to polypeptides of various lengths and amino acids. The quality of the raw material employed, their storage, and the digestion process parameters determine the quality of the peptones. Raw material must be stored under conditions that avoid growth of spoilage organisms. Fresh meat is chilled stored up to digestion, whereas frozen meat is thawed shortly before processing.

The first step in the manufacturing of peptones is the digestion or hydrolysis of the raw material. In a hydrolysis vessel the raw material is dispersed in water to which the digesting agent is added.

In the second step of the process the digest is centrifuged, so that fat and oil can be removed. Thereafter, the digest is filtered, the liquid concentrated in a vacuum heat exchanger to a syrup which is in the final step spray dried to powder.

Filtration reduces drastically the bioburden, particularly of peptic and tryptic digests that are kept over long periods at about 40 °C. After filtration the bioburden is commonly low and concentration to syrup contributes to the preservation.

The production of high quality peptones requires much more than a standardisation of the digestion process parameters. A total quality management system must be in place with emphasis on raw material specification, tracibility, non conmingly practices hygiene and cleaning and disinfection. Clearly in minimizing the risk of TSE the enzymes employed for digestion must be taken in account. They must be of non bovine origin.

The growth characteristics of peptones varies with the composition of raw materials and the digestion process parameters. Raw materials employed, such as meat or vegetables, vary in the concentration of fermentable carbohydrates. The carbohydrate concentration in the final peptone should be taking into account when assessing the growth characteristics.

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

Did you know that peptone comes from the Greek and means to digest. Peptone are a mixture of water soluble polypeptides, peptides, amino acids and other substances remaining after the digestion of protein material.

Simple proteins contain only amino acids. They produce on hydrolysis amino acids and no major other organic or inorganic products. They usually contain about 50 % carbon, 7 % hydrogen 23 % oxygen 16 % nitrogen and 0-3 % sulfur. Water soluble peptones have a Mol weight between 200 to 6000. The protein materials from which peptones are commonly produced include: bovine-, porcine-, or poultry meat, milk protein (casein), soybean, sunflower seed, gelatin, and yeast.

Peptones are termed after the origin of raw material and often the process of hydrolysis is also used. The digestion of the protein material occurs enzymatic or by acid treatment. For the enzymatic hydrolysis of protein material proteolytic enzymes such as pepsin, papain, pancreatin which contains trypsin and chymotrypsin or trypsin. The enzymes are of animal (pancreatin or pepsin) or vegetable origin (papain from papaya), or proteases from microbial origin.

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

The oxidase test is one of the most useful and common test used to differentiate groups of micro-organims. Here’s how it works:

Mode of Action
The cytochrome oxidase is an enzyme of the iron porphyrine group which is very widely distributed in nature. It oxidizes the reduced cytochrome c and is thus transformed itself into the reduced and inactive form. Through transfer of the electrons to molecular oxygen the reduced cytochrome oxidase is transformed again into the active form.

In the presence of molecular oxygen the cytochrome oxidase/cytochrome c-system can reduce a whole series of organic substances, among them the socalled NaDi reagent (1-naphthol + dimethylparaphenylene diamine) with formation of the condensation molecule indophenol blue.

This reaction is used for the classification and identification of bacteria.

Typical Composition
The reaction zone of a test-strip contains:

N,N-dimethyl-1,4-phenylene diammonium chloride 0.1 µmol; 1-naphthol 1.0 µmol.

Application
The separate colonies grown on a culture medium or, in the case of pure cultures, an inoculation loop full are being tested. Instead with bacterial mass the reaction may also be performed with a dense bacterial suspension.

Stability
See expiry date.

Only remove the amount of strips needed at the time! and do not touch the reaction zones of the test strips.

Close receptacle firmly immediately after use. The strips with deep brown coloured reaction zone are unusable. Please store at the specified temperature.

Storage
Store tightly closed in a cool dry place at +2 °C to +8 °C.

Safe removal
The test strip is to be removed safety after use like bacteria containing material. This may be done by burning, autoclaving or by placing into a 5 to 6% desinfectant solution - for at least 6 hours.

Experimental Procedure
With an inoculating loop take a separate, well-grown colony from the culture medium.

Apply the colony to the reaction zone and spread with the inoculating loop.

After approx. 20 to 60 seconds compare with the colour scale.

Evaluation
In the case of cytochrome oxidase-positive germs the reaction zone is coloured blue to blue-violet.

Medically important oxidase-positive microorganisms

Neisseria (all species), Actinobacillus ligniereslii, Aeromonas spp., Actinobacillus equuli, Pasteurella spp., Bordetella pertussis, Vibrio spp. Bac. anthracis, Cordiobacterium hominis, Bac. subtiliis
Pseudomonas spp. Brucella spp., Flavobacterium spp, Chromobacterium spp., Alcaligenes spp., Eikenella corrodens, Moraxella spp., Plesionmonas spp., Campylobacter spp. Branhamella catarrhalis
Micrococcus spp.

Oxidase-negative microorganisms
Staphylococcus spp. Pseudomonas mallei, Streptococcus spp., Pseudomonas maltophilia, Gemella haemolysans, Bordetella parapertussis, Peptococcus spp. Actinobacillus, Peptostreptococcus spp., Actinomycetem-comitans, Leuconostoc spp. Anaerobier (all), Corynebacterium spp. Haemophilus spp., Listeria spp., Pasteurella haemolytica, Lactobacillus spp. Type T, Bacillus spp., Streptobacillus
Enterobacteriaceae (all kinds), Mycoplasma spp., Acinetobacter spp., Acholeplasma spp.

Microbiology: An Introduction Media Update No Synopsis Available