Bacteria have to be grown for their identification and investigating other properties like antimicrobial susceptibility, test for enzyme production or test for virulent genes etc. By the use of appropriate method of isolation in culture medium, they can be obtained in pure cultures for further study.
Numerous and diverse type of culture medium are available today.In the time of Louis Pasteur, liquids like urine or meat broth were used as culture medium which has demerits of not being able to isolate the organism from mixed culture and hence the identification and study of other characteristics of an organism is not possible. However, liquid medium have their uses like in blood culture or bacteriological analysis of water (Most Probable Number method) when large volume of samples have to be tested and for preparing bulk cultures of bacterial antigens or vaccines.
Bacteria grow diffusely in liquid medium but produce discrete visible growth on solid medium. When inoculated in a solid medium in suitable dilutions, bacteria form colonies which are the clones of cells replicating from a single bacterium. Bacteria have distinct colony morphology and exhibit many other characteristic features like pigmentation or hemolysis which makes their identification easy. For example, On Mac Conkey medium, lactose fermenting organism forms pink colonies while non Lactose fermenting organism forms pale colored colonies.
The first person to use a solid medium was Robert Koch and he used cooked cut potato in the form of solid media. Later he used gelatin to solidify liquid media but it was unsatisfactory as gelatin is liquefied at 24oC and also by many proteolytic bacteria. The use of agar to solidify culture media was suggested by Frau Hesse, who was wife of one of the investigators in Koch’s laboratory, who had seen her mother using agar to prepare jellies.
Now days, Agar (agar-agar) is universally used in preparation of solid media. It is obtained from certain type of seaweed whose chief constituent is long chain polysaccharides with varying proportion of organic salts and small quantities of protein like substances. It has no nutritive value and is not affected by the growth of bacteria. It has a unique property that is melts at 98oC and solidifies at 42oC depending on the concentration of agar. Approximately, 1.5 to 2% agar is used for preparation of solid media. Another universal ingredient of common media is Peptone which is a complex mixture of partially digested proteins. Its constituents are proteoses, polypeptides and aminoacids, variety of inorganic salts including phosphates, potassium and magnesium and certain growth factors like Riboflavin.
Types of culture medium:
Media can be classified in many ways. On the basis of consistency concentration of agar in the media (consistency), it can be of three types. Namely:
1. Solid media (1.5-2% agar)
2. Semi solid media (0.2-0.5% agar)
3. Liquid media (no agar)
On the basis of constituent ingredients used in the media, it can be of two types. Namely
1. Simple media
2. Complex media
By the name itself, it suggests that it contains only the basic ingredients required for an organism to grow. They are generally used as general purpose media. Examples are peptone broth, nutrient broth, nutrient agar.
- Alpha hemolytic (greenish discoloration of the medium due to partial digestion of hemoglobin around the colonies)
- Beta (clear zone around the colonies )
- Gamma (no hemolysis around the colonies)
Peptone water: Peptone is a protein taken from variety of sources that has been hydrolysed to amino acids and short peptides by treatment with enzymes, acids or alkalis. When water is added to peptone, it becomes peptone water. It is commercially available.
Nutrient broth: It consists of peptone, meat extract, sodium chloride and water. It is simple basal liquid medium that supports the growth of many organisms
There are three types of nutrient broth.
a. Meat infusion broth (aqueous extract of lean meat to which peptone and NaCl is added)
b. Meat extract broth (mixture of commercial peptone and meat extract)
c. Digest broth (watery extract of lean meat that has been digested with a proteolytic enzyme so that additional peptone is not needed.)
Of these three, digest broths gives luxuriant growth of organisms and are economical.
Nutrient agar: By adding 2% agar to nutrient broth, nutrient agar can be prepared which is the simplest and most commonly used medium in routine diagnostic laboratory to grow non-fastidious organisms.
a. To detect pigment production by bacteria
b. To maintain stock cultures
c. It can be used for antimicrobial susceptibility testing (not preferably)
d. To study the biochemical characteristics of bacteria, pure growth on nutrient agar is used.
Media that contains certain ingredients of unknown chemical composition are known as Complex media. It contains special nutrients other than basal media.
These media are prepared to meet the nutritional requirements of fastidious organisms. (e.g. Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria meningitis, Hemophilus influenzae) These organisms are more exacting in nutritional needs and extra supplements like blood, serum or egg has to be added to a basal medium. e.g. Blood agar, Chocolate agar, Bordet-Gengou agar, Loffler’s serum slope, Dorsett’s egg medium
Blood agar: It is the most commonly used medium in diagnostic laboratory as many medically important bacteria are fastidious and require a richer medium than nutrient agar. It is prepared by adding 5% sheep blood to nutrient agar. It is used for isolation of fastidious bacteria like Streptococci, Pneumocooci and Haemophilus. Along with enriched media, it can also be called a differential media as bacteria can be differentiated on the basis of different types of hemolysis on the medium.
It is the color of the media rather than the ingredient from which its name is derived. Chocolate agar can be prepared by heating blood agar at 70-80OC for 10-15 minutes. It is used to grow more fastidious bacteria like Neisseria gonorrhoea, Neisseria meningitides, Hemophilus influenzae. .
Bordet-Gengou agar- used for isolation of Bordetella pertusisLoffler’s serum slope- used for isolation of Corynebacterium diptheriae
Dorsett’s egg medium – used for isolation of Mycobacterium tuberculosis and Corynebacterium diptheriae
It is a liquid medium to which inhibitory substances that inhibits the growth of unwanted bacteria and favors the growth of wanted bacteria. This medium provides nutrients and environmental conditions that favor the growth of a particular bacterium but not others and hence there is absolute increase of the wanted bacterium relative to other bacteria.
It is generally used in mixed cultures or in materials containing more than one type of bacteria (e.g. stool culture) where the unwanted bacteria (non pathogens or commensals) can overgrow the one that is to be isolated (pathogenic). For example, Salmonella Typhi can be overgrown by Escherichia coli in culture from faeces if enrichment media is not used. e.g. Tetrathionate broth, Selenite F(Feces) broth, Alkaline peptone water.
Tetrathionate broth: It inhibits coliforms allowing typhoid and paratyphoid bacilli to grow freely in fecal sample Selenite F (Feces) broth: It is used for Shigella sps in fecal sample (dysentery)
Alkaline peptone water: It is used for Vibrio cholerae.
Selective media: When a substance that is inhibitory to unwanted bacteria and favors the growth of wanted bacteria is added to a solid medium, that medium is known as Selective media. Inhibitory substances might be bile salts, dyes, acids, alcohols and antibiotics. These media are used to isolate a particular bacterium from a sample with mixed bacteria flora.
This is a type of medium inwhich an inhibitory substance is added to favor the growth of a particular bacteria of interest while inhibiting the growth of other unwanted bacteria. Inhibitory substance may be dyes, bile salts, alcohol, acids and antibiotics. These medium are used to isolate a particular bacteria from a specimen expected to have mixed bacterial flora. e.g.
- Deoxycholate citrate agar (DCA) – deoxycholate in the medium acts a selective agent in isolating Shigella sps
- Wilson Blair’s brilliant green bismuth sulfite agar – used for isolation of Salmonella sps
- Lowenstein Jensen medium (LJ) – used for isolation of Mycobacterium tuberculosis
- Thayer-Martin medium – used for isolation of Neisseria gonorrhoeae and Nesseria meningitidis
- Potassium tellurite medium – used for isolation of Corynebacterium diptheriae
- Thiosulphate Citrate Bile salts Sucrose agar (TCBS) – used for isolation of Vibrio cholerae
- Mannitol Salt agar – used for isolation of Staphylococcus aureus
- Mac Conkey agar- used for isolation of gram negative bacilli and inhibits the growth of gram positive bacteria.
A medium in which certain indicator is or reducing subtance is added and growth of bacteria is indicated by change in color of indicator used in the medium is known as Indicator medium.e.g.
- Mac Conkey agar – Lactose present in the medium is fermented by lactose fermenters as acidic pH produced during fermentation changes the color of neutral red indicator to produce pink colonies while non lactose fermenters produce pale colonies.
- Wilson Blair medium- contains sulfite as an ingredient. Salmonella typhi reduces sulfite to sulphide in presence of glucose imparting bacterial colonies with black metallic sheen
A medium in which a substance is added that bring out differing characteristics of bacteria thus enabling to distinguish between them is known as a Differential medium.e.g.
- Blood Agar: differentiates between different bacteria on the basis of lysis of blood cells in the medium
- α- haemolysis – greenish discoloration around the colonies due to partial degradation of hemoglobin – Streptococcus pneumoniae, Viridans Streptococci
- β- haemolysis – clear zone around the colonies due to complete lysis of the red blood cells – Staphylococcus aureus, Streptococcus pyogenes
- γ- haemolysis – no haemolysis at all. Enterococcus fecalis
- Mac Conkey agar: differentiates between Lactose fermenters (pink colonies) and Non lactose fermenters (pale colonies)
- Lactose fermenter- Escherichia coli, Klebsiellasps
- Non Lactose fermenter- Proteus sps, Salmonella sps, Shigella sps
- Late Lactose fermenter- Shigella sonnei
Sugar media consists of 1% sugar in peptone water along with an indicator (Andrade’s indicator- 0.05% acid fuchsin in NaOH). A small tube (durham’s tube) is kept inverted in the sugar tube to detect gas production. Initially, colour of the medium is light yellow and if the organism ferments sugar, acid is produced which is indicated by the change of colour to pink. If gas is produced, it is accumulated in the durham’s tube.It is used for characterisation and identification of bacteria.Sugar may be any fermentable substance like
- Monosachharides: Pentose – arabinose, xylose, rhamnose
- Hexose – glucose, fructose, galactose, mannose
- Disachharides: sucrose, maltose, lactose, trehalose, cellibiose
- Trisachharides: raffinose
- Polysachharides: starch, inulin, dextrin, glycogen
- Polyhydric alcohols: glycerol, erythritol, adonitol, mannitol, dulcitol, sorbitol, inositol
- Glycosides: salicin, aesculin
- Organisc acids: tartarate, citrate, gluconate, malonate
Among these glucose, sucrose, lactose, mannitol are routinely used for sugar fermentation test.
Hiss serum sugars are used for certain fastidious organisms like Pneumococci, Gonococci and Meningococci that reqiure serum for growth. It consists of 3% serum in distilled water.
A transport media is a holding medium designed to preserve the viability of organism in the specimen but does not allow multiplication.When specimens like throat swab, uretheral swab etc. that are likely to contain fastidious organsims (Gonococci) and any delay in transport to the laboratory is anticipated, transport media is used to make sure that they will survive during the transport and won’t be overgrown by nonpathogens.e.g.
- Stuart’s transport medium and Amies transport medium – Neisseria gonorrhoeae
- Cary Blair medium- Vibrio cholerae, Campylobacter sps
- Alkaline Pepetone water (pH 8.6) – Vibrio cholerae
These medium are used to grow anaerobic organisms and contain reducing substances.e.g. Thioglycolate broth, Robertson’s cooked meat medium
It contains reducing substances such as sodium thioglycolate, glucose, ascorbic acid, cystiene and agar (0.1%) with methylene blue. Thioglycolate acts as a reducing agent and creates anaerobic environment deeper in the tube allowing anerobes to grow. Agar prevents convection currents of air. Methylene blue acts as an redox potential indicator which shows that that the medium is anaerobic except the surface layer.
Robertson’s cooked meat medium:
This medium consists of meat infusion or nutrient broth with cooked minced meat of beef heart. Liquid parrafin is layered over broth to prevent entry of air. Unsaturated fatty acids in the minced meat act as a reduicng agent and absorb oxygen. Sulphahydryl groups present in aminoacid cystiene and glutathione create a low redox potential. The specimen is inoculated deep in the medium in contact with the meat particles.
It also detects proteolytic and saccharolytic activities of anaerobes. Proteolytic anarobes blacken the meat particles with the formation of foul smelling sulfur compounds. Sachharolytic anaerobes turn meat red or pink with sour smell.
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