Many, microorganisms can be cultured either in liquid or on agar based solid medium. A culture is a collection of
microbial cells growing in or on a medium. A culture medium may be defined in general as any material which
supports the growth/replication of microorganisms.
In Microbiology, a media is a solid or liquid preparation containing nutrients for the growth of microorganisms.
There is always a need to culture and grow microorganisms under more or less well defined conditions. A suitable
nutrient medium to provide the components for growth is therefore necessary, as well as sterile technique, to maintain the culture in pure form. The important benefit of a solid medium is that an individual cell inoculated onto the surface can develop to form a visible colony which forms the basis
of most microbial isolation and purification methods.
Microbiological media are used for the growth or culture of microorganisms, diagnostic (identification) tests, storage and transportation of microorganisms, growth or maintenance of cells in a tissue culture.
TYPES OF MEDIA
Three kinds of media are described based largely on their composition, as natural media, semisynthetic media and synthetic media.
1. Natural Media:
Natural media consists of natural
basic materials such as oat, corn meal, yeast extract etc. An disadvantage of natural media is that their exact
composition is not precisely known. Hence they are not suitable for precise metabolic investigations.
Examples include hemp seed, cornmeal agar, apple juice agar, Oatmeal, potato dextrose agar (PDA), malt extract agar etc.
2. Synthetic Media:
These are media whose chemical
composition is precisely known and used for specific investigations. An example is the Czapeks solution: CNaNO3, KHPO4, MgSO4, 7H,OKCI, FeSO4: 7H;0, Sucrose and distilled water)
3. Semi-Synthetic media:
These are media in which one
or all its components can be duplicated exactly. Semi synthetic media can be produced from Czapeks agar by adding malt extract. It is semi-synthetic because
we do not know the exact composition of the malt extract.
TYPES OF ISOLATION MEDIA
There are several types of primary isolation media:
1.General Purpose Media:
These are non-selective primary isolation media used for culturing a wide
of fastidious variety Actinonyces, Brucella, Clostridium, Enterobacteriaceae, microorganisms including Neisseria, Pseudononas, Streptococcus. Often these formulations are enriched Staphylococcus
with materials such as bIood, haemoglobin, sera or and coenzyme haematin (X factor).
2. Selective Media:
Selective media have been modified in some manner to suppress or prevent the growth of one group of organisms while permitting the growth of another group from a mixed flora.
The modification of the medium may be made by altering the pH to take advantage of the ability of some
organisms to grow at one PH and not at another. Another principle invoked in the selection of a particular type of organism from a mixed population
is to utilize the inhibitory (bacteriostatic) property of a specific chemical without which the medium would be suitable for many species in the sample.
For example, addition of crystal violet in a final concentration 1:100,000 will inhibit most gram positive types without affecting the gram-negative group. Similarly, antibiotics may be used for selective inhibition for example sodium azide (0.03%) has bacteriostatic action against gram-negative bacteria,
the aerobic gram-positive spore forming bacilli, and certain other aerobes. The degree of selectivity may also be chosen. Moderately selective media inhibit
unwanted organisms to the point where they do not interfere with growth of wanted organisms.
Highly selective media, on the other hand, strongly block the growth to completely eliminate undersirable cultures.
Examples are Brilliant Green agar, Desoxycholate citrate agar, MacConkey agar, and Xylose Lactose Dextrose (XLD) agar.
3. Enrichment Media:
Enrichment media suppress the
growth of competitive normal flora while enhancing the growth of the desired culture. Specimens known to be highly contaminated with normal flora often require exposure to enrichment media to be able to isolate the primary pathogen
present Examples include Tetrathionate broth base, selenite-F broth,
Brain Heart infusion, Buffered peptone water. An Enrichment medium is in liquid form whereas an Enriched medium is a solid medium. Enrichment
media are often used as transport media.
Specialized isolation media:
This group includes those formulations that satisfy nutritional needs of specific groups of organisms. These group of organisms grow in the specialized isolation media forming characteristic colonies that makes it easy for them to be differentiated and identified.
5.Differential media:
Bacteria display all possible variations in their nutritional requirements and advantage can be made of this fact for identification.
Some media contain one or more fermentable carbohydrates and a suitable indicator for the detection of acid or alkali production; some contain
substance rich in sulphur and an indicator to detect hydrogen sulplide formation. Examples of differential
media include MacConkey agar, Desoxycholate Citrate agar.
6. Selective differential media:
Some media combine the characteristics of being selective as well as differential toward the isolation of various microorganisms. Some media when used
for particular microorganisms exhibit these attributes.
The following media when used for Salmonella are regarded as selective differential media: Bismuth sulplite agar, XLD agar, Hektoen enteric agar, MacConkey agar, Brilliant Green agar, Salmonella – Shigella agar (SSA), Desoxycholate agar and Desoxycholate citrate agar, Phenylethanol agar and Columbia nutrient agar.
7. Selective enrichment media:
These media contain compounds that enrich some organisms thereby
selecting them for easy isolation. Such media include selenite cystine broth, tetrathionate broth, lauryl tryptose broth.
8. Transport media:
Transport media are essentially non
nutrient, semi-solid, highly reductive media which inhibit self destructive enzymatic reactions within the
cells. They also prevent the lethal effects of oxidation, thus maintaining the organisms present at a status quo
condition.
Among the widely used transport media are Stuart and Bacto transport medium. Others are
Remel Carry Blair transport medium, charcoal cephalexin blood agar (CCBA) and Bordetella transport medium, Glycerol saline transport media
Virus transport medium (VTM).
9. One purpose medium:
These are media used for a particular type of organism. It is normally used for specific genus or species of microorganisms.
Examples of these media include Nitrogen free malate (NEM) stock solution media for Azospirillum, Monsur’s Tellurite Taurocholate gelatin agar for Vibrio cholerae, Thiosulphate citrate bile sucrose (TCBS) agar for vibrios, yeast peptone (YP) medium
for Bdellovibrios, Pringsherim soil medium for Spirillum volutans, Giesberger’s base medium for
Aquaspirillum and Oceanospirillıum, Fletcher’s agar, Elling-husen and McCullough (EM) medium and
modified Rortloff’s medium for leptospira BSK 11 medium for Borrelia burdgoferi, Glucose yeast peptone
tryptone, Glucose tryptone yeast and SZ medium for Spirochaeta litoralis.
10. Microbiological assay media:
Specific microorganisms can be used to measure the concentration of substances
such as antibiotics and vitamins. For instance, blood serum or other tissue fluids can be assayed for antibiotics by using microorganisms known to be
susceptible to those antibiotics.
This type of assay involves the measurement of growth inhibition caused by the antibiotic within established limits. The degree of inhibition is proportional to the amount of drug. One of the assay techniques uses wells made in the bacteria-containing agar medium that are filled with sample fluid. As the fluid diffuses out of the
wells, zones of microbial inhibition are visible.
Microbial assay of antibiotics are also made on pharmaceutical product, animal feeds and other materials.
Microbial isolates from clinical specimen are routinely tested for their agents. The results of one susceptibility to selected antimicrobial assay of this type is called the minimum inhibitory concentration (MIC) which is the lowest concentration of the test agent to inhibit growth of
the microorganisms.
Results from various sensitivity tests
guide the physician in selecting appropriate treatments for
infected patients. Standardized microbiological assay media
for different chemical agents are commercially available, an
example is the Mueller Hinton agar, Sensitivity Tests Agar (STA).
PREPARATION OF CULTURE MEDIA
For any medium to be used for the isolation of microorganisms, it must be prepared in such a way that it will not give false results.
Sterilization of media can be achieved by autoclaving after preparation of the media according to manufacturer’s specifications. Test for sterility
is usually required and can be achieved by incubating the prepared media for a period of 18 to 24 hours and then examine for microbial growth.
Sterilization of media can be done at
high temperature (short time) or low temperature (long time) depending on the type of media. In order to prepare
culture media correctly, a manual should be prepared which gives the exact preparation of all media used in the
laboratory. It should also include pH testing, sterilization, storage, shelf life, performance testing and a brief
description of the ready- medium (e.g colour and opacity) and its use.
CHOICE OF CULTURE MEDIA TO USE FOR ISOLATION
The selection of culture media to use for the isolation of any microorganisms will depend on the requirement of the organisms and features by which they are recognized, whether the specimen being cultured are from sterile sites or from sites having normal microbial flora.
Although a selective medium is usually more expensive than a non selective one, the use of a selective medium often avoids sub culturing and isolates an organism more quickly. It makes it easier to differerntiate and interpret the growth of the microorganisms especially by laboratory staff and students with limited experience.
HOW TO POUR MEDIA
1. Collect containers of Nutrient agar, Mac Conkey agar and Saboraud Dextrose agar from you instructor.
2. Read the manufacturers instruction carefully
3. Using ratio, calculate the quantity of each agar for the preparation of 200mls (10 plates).
4. Dissolve accordingly in separate flasks.
5. Sterilize by autoclaving
6. Allow the molten agar to cool to about 50°C (just cool enough for comfortable handling after removal from autocalve). Unscrew cap of bottle with little finger. It can be held by the little finger during the procedure to avoid placing it on the bench where it could become contaminated.
7. Flame mouth of bottle for a few seconds. This is done to produce an upward flow of air from the bottle so
that any organisms in the area will not fall into the bottle. (It is not done to kill microorganisms – the bottle would have to be heated for too long to achieve
this effect).
8. Pour the molten agar (20cm) slowly into the base of the sterile petri dish, lifting the lid only as much as necessary. Do not spill the agar on the edges of tho
dish. Replace lid and allow agar to set.
Note: Watch your instructor or demonstrator pour some
plates before you practise.
9. Incubate your plates for 18-24hrs (for sterility check).
10. Observe and report on the sterility of your plates.