One from Virginia related to the use of TCBS agar to grow Vibrio cholerae, so I thought that this week I would cover some “old fashioned” microbiology and talk about agar. Really! I hear you cry “that’s not very interesting”! Well, as “interesting” is banned when discussing microbiology, I’ll use “actually very clever” to describe the reasons why we use different types of agar in the laboratory… here goes…
It is the added chemicals and compounds that dictate what bacteria will grow and how they will appear on the agar. Agar plates can be divided into:
- Selective PLUS indicator
Non-selective agar is chosen to grow commonly isolated bacteria, in particular when the laboratory is not expecting there to be a mixture of different types of bacteria and where the target bacterium is sufficiently hardy to grow with the minimum amount of support. An example of a non-selective agar is Nutrient Agar (NA). NA contains the basic components for common bacteria and fungi to survive; an energy and nutrition source (e.g. beef or yeast extract), water, physiological amounts of electrolytes (e.g. sodium) and some nitrogen. NA agar will easily grow bacteria such as Staphylococcus aureus, Escherichia coli, Beta-haemolytic streptococci, Candida spp., etc.
The most common use of non-selective agar in the laboratory is to keep something growing in pure culture for further tests e.g. for short term storage in case further results are required at a later date or in order to send the sample away to a reference laboratory for further antibiotic sensitivities or identification.
Selective agar has additional chemicals or compounds added to try and STOP non-target bacteria and fungi growing. These types of media are used when the laboratory is expecting a mixture of bacteria to grow but they are only interested in growing one specific bacterium or a limited number of bacteria. The selective nature of the media stops the background bacteria growing, making it easier to spot the target bacterium. The background bacteria are often “normal flora”, which are not causing infection.
Examples of selective agar include Modified New York City (MNYC) agar, Lowenstein-Jensen (LJ) media and Sabouraud agar.
- MNYC contains antibiotics such as Vancomycin and Colistin and is used to isolate Neisseria gonorrhoea from genital specimens. Vancomycin suppresses the growth of most Gram-positive bacteria while the Colistin suppresses the growth of Gram-negative bacilli, whereas N. gonorrhoea can grow in the presence of both these antibiotics. The selective inhibition of bacteria means that the normal flora of the genital tract, which might include Enterococcus spp., Enterobacteriaceae and skin bacteria, do not overgrow the agar plate and hide the N. gonorrhoea.
- LJ media is used to grow Mycobacteria spp. LJ media has lots of different components but malachite green, low levels of penicillin and Nalidixic acid are all added to inhibit the growth of other bacteria. This inhibition is essential as most Mycobacteria spp. are very slow growing (some can take as long as 12 weeks!) therefore other bacteria would easily outgrow them.
- Sabouraud agar is used to grow fungi including yeasts and moulds. This agar has an acidic pH (about pH 5.6) which stops bacteria growing. Most moulds are slow growing (potentially up to weeks) and would be overgrown with bacteria unless these were suppressed.
Fastidious agar contains additional nutrients to HELP GROW bacteria which have specific growth requirements. Some bacteria are just too weedy to grow on even non-selective media and require extra help, just like adding fertiliser to plants (remember fastidious by “fuss”tidious agar, it gives fussy bacteria extra supplements to help them grow). Examples of fastidious agar include chocolate blood agar and fastidious anaerobe agar (FAA).
- Chocolate blood agar sadly does not actually contain chocolate; I was gutted when I first learnt this but life is full of these little disappointments! Chocolate agar is so called as it contains chocolatized blood; it contains blood that has been heated to break it down and make the nutrients contained in the blood more readily available (it appears a nice chocolate brown colour as well!). For example, Haemophilus influenzae requires NAD (Nicotinamide adenine dinucleotide also known as Factor V) and haemin (Factor X) to grow. Haemophilus influenzae will not grow on normal blood agar as it is fastidious, it requires specific growth requirements.
- FAA contains additional cysteine and peptone amongst other chemicals and compounds which help anaerobes to grow, which have very specific growth requirements. Although it is usually incubated anaerobically FAA can also be incubated aerobically and then be used to help fastidious aerobes to grow (e.g. Abiotrophia defectiva, sometimes known as nutritionally variant streptococcus, needs the extra cysteine but prefers the aerobic environment).
Indicator agar contains a chemical or compound which reacts in a certain way in the presence of a specific bacterium. This reaction allows the laboratory staff to start to identify the bacterium growing. Examples of indicator media include Blood agar and Cysteine lactose electrolyte deficient (CLED) agar; both of which are used a lot in microbiology laboratories.
- Blood agar is a nutrient agar which contains sheep’s blood. In the distant past it used to contain horse blood or even human blood! The blood in the agar plate indicates whether the bacteria growing can haemolyse the blood or not. The type of haemolysis then helps identify the type of bacterium e.g. Beta-haemolytic streptococci cause complete (beta) haemolysis whereas viridans streptococci cause incomplete (alpha) haemolysis which looks green (hence viridans from the latin, viridis, for green).
- CLED agar will grow most commonly isolated bacteria but its main use is to identify bacteria which ferment lactose. Bacteria which ferment lactose such as E. coli produce an acid which turns the agar yellow, by reacting to a pH indicator, whereas bacteria that don’t ferment lactose remain blue. The lack of electrolytes in the agar also stops the lactose non-fermenter Proteus mirabilis from swarming all over the plate and obscuring other bacteria. It doesn’t stop P. mirabilis growing so it is not selective; it just stops it being unruly.
Most agars in common use in microbiology laboratories are both selective and indicator. They contain chemicals and compounds that suppress non-target bacteria as well as help identify those bacteria which do grow. Examples of these types of agar include MacConkey agar, Xylose lysine deoxycholate (XLD) agar as well as the Thiosulphate citrate bile salt (TCBS) agar, which I mentioned last week for growing Vibrio cholerae.
- MacConkey agar is used to grow gut bacteria from mixtures because it contains bile salts which inhibit the growth of most other types of bacteria. MacConkey agar also contains lactose and a pH indicator which means that lactose fermenting bacteria cause a deep red colour and this helps with the initial identification of the bacterium (as for CLED agar above). The agar is SELECTING gut bacteria and INDICATING their ability to ferment lactose.
- XLD agar is used to look for bacteria that cause gastroenteritis, especially Shigella spp. and Salmonella spp. Like MacConkey, XLD contains bile salts to suppress any bacteria not normally grown in the gut. XLD also contains a pH indicator but the XLD agar itself is also alkaline and so the agar normally looks pink. Most gut bacteria ferment the xylose sugar in the agar and produce yellow colonies whereas Shigella spp. don’t so the colonies remain pink. This allows the laboratory staff to rapidly spot potential Shigella spp. colonies. Salmonella spp. ferment xylose and the agar turns yellow, although once the xylose has all been used up, by the bacteria’s growth, the colonies go pink again. However Salmonella spp. also metabolise thiosulphate in the agar to produce hydrogen sulphide which makes the colonies go black. Salmonella spp. can therefore be easily distinguished from most other bacteria growing on the plate. This kind of agar allows the laboratory staff to rapidly look at the XLD plate and see if anything like Shigella spp. or Salmonella spp. are growing and if not put the plate back down and forget about anything else that might be growing.
- TCBS contains sodium thiosulphate, sodium citrate and bile salts which stop almost all other bacteria except Vibrio spp. from growing, it is therefore very selective. This selection is enhanced by the high pH of the agar which stops other bacteria growing whilst enhancing the growth of Vibrio spp. TCBS also contains sucrose and an indicator media, a colour change mechanism which can indicate if the bacteria growing can ferment sucrose. V. cholerae goes yellow on TCBS as it ferments sucrose whereas V. parahaemolyticus stays green as it does not ferment sucrose.
- Help them select out certain types of bacteria and fungi
- Enhance the growth of those which are a bit fussy or “fustidious” I mean fastidious in their growth requirements
- Indicate which bacteria need further follow up
I think agar is really rather clever. If you agree then why not become a Biomedical Scientist? It’s a challenging career choice, there’s a lot to know but a great choice if you are scientific. If all of this old-fashioned microbiology rambling is too much, not to worry at least you’ve now got to the end of the blog… and can find something more “interesting” to do…!