Saturday, September 25, 2021

Culture Media Part 4- Classification Based on Function

Based on the application or function media can be divided into Supportive media (General purpose media) and special media. Special media includes a number of media like Enriched media, Enrichment media, Selective media, Differential media, Indicator media, Sugar media and Transport media. However, sometimes a single media may fulfil more than one functions. For example; Blood agar can act as both Enriched and Differential media. Eosin Methylene Blue (EMB) agar can act as both Selective and Differential media. MacConkey (MAC) agar can act as Selective, Indicator and Differential media. Mannitol Salt agar can act as both Selective and Differential media. 1. Supportive media (General purpose media) This media support the growth of most of the organisms and do not contain any added inhibitors. Basal media used in microbiology lab like Peptone water, Nutrient Agar and Nutrient Broth can be considered as supportive media for growing non-fastidious organisms. Media such as Tryptic Soy Broth and Tryptic Soy Agar are also examples of GPM as they sustain the growth of many microorganisms. Blood or other nutrients can be added to the GPM to support the growth of fastidious organisms, then they are called Enriched media. 2. Special Media 2.1. Enriched Media Enriched media contains special ingredients like blood, serum or egg added to the basal media. It is used to cultivate microorganisms that are more exacting in their nutritional needs.ie; to grow fastidious organisms. Eg: Blood agar, Chocolate agar, Egg media and Brain Heart Infusion Broth. a) Blood agar:

Blood agar contains 5% mammalian blood added to the basal media. Blood is added after autoclaving the media. It is enriched medium as it contains many nutrients like protein, carbohydrate, lipid, iron, a number of growth factors and vitamins that can support the growth of many fastidious organisms including aerobic and anaerobic bacteria. Vitamin K, cysteine and hemin in blood support the growth of anaerobic organisms. Blood agar acts as both enriched and differential medium. Blood agar is a differential media as it distinguishes haemolytic and non- haemolytic bacteria. Hemolytic organisms produce hemolysin, a protein that cause hemolysis or breakdown of Red Blood Cells (RBC). Sheep blood is preferred as hemolysis is more clearly defined on it.  Hemolysis appears as a clear zone if there is complete lysis of RBC (β hemolysis) or greenish halo around the colony if there is partial lysis of RBC (α hemolysis). Blood agar also help to differentiate among Gram positive cocci.

Eg: Streptococcus pyogenes and Staphylococcus aureus cause β hemolysis.  Streptococcus viridans cause α hemolysis.

a)                 Chocolate Agar: Used to grow fastidious organisms like Neisseria gonorrhoeae, N. meningitidis, H. influenza and Pneumococci. It is called chocolate agar due to its chocolate brown appearance. The brown colour is the result of heating red blood cells and lysing them before adding to the media. Heating a mixture of sheep blood and nutrient agar releases haemoglobin, hemin or X factor and Nicotinamide adenine dinucleotide (NAD or V factor) which gives the brown colour. 

b)                 Brain heart infusion broth: It is highly nutritious buffered liquid media for cultivating fastidious organisms. It is prepared using non- enzymatic infusion from calf brain and cow heart with added peptone and dextrose.

1.1.  Enrichment Media

In samples containing mixed population of microorganisms the bacteria to be isolated is often overgrown by the unwanted microbes.  Usually non-pathogenic or commensal bacteria tend to overgrow the pathogenic ones. Enrichment media are liquid media containing special nutrients that stimulate the growth of particular organisms that might not be otherwise present in sufficient amount for isolation and identification. For eg: In fecal samples S typhi may not be sufficiently numerous for identification and usually overgrown by E coli. If cultivated on the medium Selenite cystine (SC) broth with trace element selenium greater number of S typhi could be obtained on incubation and selenium is inhibitory to coliforms. Such media which contain substances that have a stimulatory effect on wanted organisms are called enrichment media. Other Egs: Tetrathionate broth to promote growth of Typhoid and Paratyphoid bacilli in samples containing coliforms; Selenite F Broth for Dysentery bacilli.     

1.2.           Selective media

Selective media are solid media that contain nutrients to promote the growth of wanted organisms and also contain substances that inhibit or suppress the growth of unwanted organisms. Incorporation of substances like bile salts or dyes such as basic fuchsin and crystal violet allow the growth of Gram negative organisms while they inhibit the Gram positive ones. MacConkey agar, Eosin Methylene Blue Agar are examples of selective media which are widely used for detection of E coli in water supplies. These media will not allow the growth of Gram positive organisms.

a) MacConkey Agar is made up of peptone, lactose, agar, neutral red, taurocholate, crystal violet and bile salts. Crystal violet and bile salts act as selective agents and inhibit the growth of  G +ive bacteria and allow G –ive growth.

b) Eosin Methylene Blue Agar contains two dyes, Eosin Y and Methylene Blue that inhibit G +ive bacteria.      

c) Mannitol Salt Agar allows the selective growth of halophiles. 7.5% NaCl act as selective agent promoting the growth of halophiles and inhibiting non-halophiles.                                                                                                                           

d) Sulfadiazine and Polymyxin Sulfate (SPS) Agar: It is used to isolate Clostridium botulinum which causes food poisoning. This media promote the growth of  Clostridium botulinum while inhibiting the growth of most other Clostridium species.  It contains the antibiotics sulfadiazine and Polymyxin sulphate as selective agents.

e) Desoxycholate Citrate Agar for isolating Dysentery bacilli

f) Thiosulphate Citrate Bile Sucrose Agar (TCBS) for isolation of Vibrio species from fecal samples. Alkaline pH of the medium promote the growth of Vibrio species and high concentrations of sodium thiosulfate and sodium citrate to inhibit the growth of Enterobacteriaceae.

g) Thayer Martin Media used to isolate N.gonorrhoeae from clinical specimens contain antimicrobials like vancomycin, colistin, nystanin in Chocolate agar. On incubation in the presence of 3-10% CO2, this media will give colonies of N.gonorrhoeae and inhibit other commensals.

h) Lowenstein-Jensen Media used for the isolation of Mycobacterium species. This media contains mineral salts, asparagine, glycerol, malachite green and hen’s egg.  Malachite green in the medium act as the inhibitor for unwanted organisms.

1.3.           Indicator media

Indicator media contains an indicator that changes its colour when bacterium grows in them. Blood agar can be considered as an indicator media. In some indicator media, a pH indicator will be incorporated. As the organisms grow, pH of the medium will be changed due to acid production and it causes the indicator to change its colour. Example is MacConkey agar. The presence of the pH indicator neutral red makes it an indicator media. The indicator changes the colonies of lactose fermenters into red or deep pink in colour and leaves the non-fermenters colourless or translucent. Lactose fermenters catabolise lactose by fermenting it and release acidic waste products. These acidic waste diffuse into the media, changing the colour of indicator. Neutral indicator is red or orange in acidic pH (<6.8) and colourless when pH is over 6.8. The indicator imparts pink or red colour to colonies of the lactose fermenters.  

Other Examples:

Wilson & Blair Medium for isolation of S typhi. This media contains the indicator Sulphite. S typhi reduces Sulphite to Sulphide in the presence of Glucose and produce colonies with black metallic sheen.

McLeod’s Medium or Potassium Tellurite Agar used to isolate C.diphtheriae. Potassium Tellurite in the medium is reduced to metallic tellurium by C.diphtheriae to produce black colonies.

Thiosulphate Citrate Bile Sucrose Agar (TCBS) for isolation of Vibrio species. Bromothymol blue is the indicator in the medium (Yellow in acidic, green in neutral and blue in alkaline conditions). Organisms that ferment sucrose appear as yellow colonies while the non-fermenters appear as green colonies. Hence act as an indicator medium also.

1.4.           Differential media

Differential media helps to distinguish among different groups of microorganisms and even permit identification of organisms based on their biological characteristics. In some cases, indicator in the differential media changes its colour when a particular biochemical reaction occurs. Many of the media we mentioned yet also falls under differential media.

Blood agar which is an enriched media, indicator media, is an example of differential media too. It helps to differentiate haemolytic and non-haemolytic organisms. ie; they differentiate organisms based on their ability to produce hemolysin.   MacConkey agar is also a differential media as it helps to differentiate between lactose fermenters and non-lactose fermenters. Neutral red indicator changes its colour in acidic pH and impart red colour to lactose fermenters. Most of the commensals of intestine including E coli are good lactose fermenters and appear pink to red in colour whereas most of the pathogens like Salmonella and Shigella are non-lactose fermenters and appear colourless.

Mannitol salt agar, selective media for halophiles act as differential media too. It helps to differentiate pathogenic and non-pathogenic Staphylococci.  Pathogenic ones like Staphylococcus aureus release acidic by-products as they make use of mannitol as carbon or energy source.  Acidic products cause the phenol red indicator in the medium to change into yellow colour (Phenol red indicator: Yellow in acidic condition and red in alkaline conditions).

EMB agar which is a selective media also serve as the differential media. Eosin Y and Methylene blue dyes in EMB agar react with acidic products released by some G –ive bacteria when they use lactose or sucrose in the medium as carbon and energy sources. Fecal bacteria such as E coli produce large amounts of acidic products and have green metallic sheen on EMB agar. Help to differentiate E coli.

SPS (Sulfite Polymyxin Sulfadizine) Agar, selective media for Clostridium botulinum also serve as differential media. Clostridium botulinum produce black colonies on SPS Agar because of the production of hydrogen sulphide by the organism from the sulphur containing additives. 

1.5.           Sugar media

Sugar media contains fermentable sugars in it. It can be monosaccharides like pentoses (eg: Arabinose, Xylose) or hexoses (eg: Dextrose, mannose), disaccharides (eg: Saccharose, lactose), trisaccharides (eg: Raffinose) or polysaccharides (eg Starch). Sugar media has two functions. Primary function is that the media will act as readily available source of energy, provided the organisms are able to utilize the sugar present in the media. Second function is that it is helpful in the identification and classification of organisms.

Sugar media consists of 1% sugar in peptone water along with an indicator. Durham tube is kept inverted in the media to detect gas production. Indicator changes its colour if the inoculated organism ferments the sugar. Fermentation could be with or without gas production. Gas production is indicated as bubble formation in Durham tube. 

1.6.           Transport media

Special media devised to transport the microorganisms are called transport media. They are usually used to transport either fastidious or delicate organisms that cannot withstand the time taken for transport of specimen from the site of collection to the laboratory. Transport media are essentially buffered solutions containing carbohydrates, peptones and other nutrients (excluding the growth factors) which would preserve the viability of bacteria during transport without allowing their multiplication. Examples: Glycerol saline for Enteric bacilli, Stuart’s medium for Gonococci, Venkatraman Ramakrishnan (VR) medium for Vibrio cholera.

 

Examples of  multipurpose media

Blood Agar

Enriched /indicator/ differential media

MacConkey agar

Selective/ indicator/ differential media

Eosin Methylene Blue Agar

Selective /differential media

Mannitol Salt Agar

Selective /differential media

Sulfadiazine and Polymyxin Sulfate (SPS) Agar

Selective/ differential media

Thiosulphate Citrate Bile Sucrose Agar (TCBS)

Selective/ indicator media


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