MEDIA FOR ISOLATION AND IDENTIFICATION

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MacConky Agar

These media composed of;

Sodium chloride

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Lactose

Neutral red

Agar

Peptone

Sodium taucholate

The media should be prepared via manufacturer instruction.

These media is used for differentiating intestinal organism into pathogen and non pathogenic fermenter micro organism, the nutrient substance of these media is peptone while agar is a solidifying agent, sodium taurocholate is bile salt agent that inhibits the growth of several gram positive organisms, and the lactose served as lactose fermenting organism, while neutral red used as indicator. The indicator work by the process whereby when the fermentation take place it yield acid and these acid react with bile salt to give red color, while non lactose fermenter exhibit alkaline reaction in these case will absorb neutral red and give a colorless colonies, but if MacConkey agar is prepared without sodium chloride been added, the media will provide electrolyte. The present of low electrolyte it resists most proteus species from covering the surfaces of the media.

Deoxycholate Citrate Agar

These media composed of;

Agar

Neutral red

Meat extract

Sodium deoxycholate

Lactose

Proteose peptone

Ferric citrate

Sodium citrate

Sodium thiosulphate

Preparation strictly on manufacturer instruction

These media is used as a selective media for examination and identification of shigellae and salmonellae species. Neutral red is used as an indicator, while sodium deoxycholate (bile salt) serve as an inhibiting agent, the substance inhibit the growth of several gram-positive organisms, and permits the growth of intestinal gram-negative however sodium deoxyholate is a poisonous substance in the present of sodium citrate, and sodium thiosulphate is also poisonous to coliorms, also some degree in salmonellae species, but this poisonous substance are render non poisonous by addition of ferric citrate, these substance that that render the media non poisonous only confer to salmonellae species, it does not take effect on coliform micro organism. The reason why coliform micro organism doe not growth on these media effectively is because they do not grow as of that of non lactose fermenters, the growth of coliform yield pinks color of colony because of the acid produce round the colony of the species cause the sodium deoxycholate to separate from the colonies which permit the organism to growth effectively. The case of proteus species it show a colorless and the colony remain in cluster without spreading, and a small black dot appears in the center of the cluster colonies, and poses odour like that of fish. Colorless colonies also show by shigellae and salmonellae species, but in some case of shigellae when left for sometimes it later develop a pink color doe to ingestion of lactose fermentation by the organism. Doe to fermentation of thiosuphate (H2S) reaction with ferric citrate in the media to form ion sulphate, this reaction result in formation of black dot in the center of salmonellae colonies in the media.

Cystine lactose electrolyte deficiency (CLED): these media also called mackey and sandy.

Composition

Peptone

Bromthymol blue agar

Meat extract

Tryptone

Lactose

L-cystine

Preparation is base on the manufacturer instruction

The media is strictly used for cultivation of micro organism from urine specimen. Doe to deficiency of electrolyte in the media, it inhibits the swimming around of proteous species and give good differentiation of the pathogen obtained from the urine.

Wilson and Blairs media: these are media that are used for isolation of salmonellae typhi and paratyphi.

Composition

Sodium phosphate crystal

Nutrient agar

Brilliant green

Dextrose anhydrous sodium sulphate

Ferric citrate

Bismuth ammonium citrate

Preparation is base on the manufacturer instruction.

Brilliant green in the media serve as an indicator agent, the substance inhibit the growth of E. coli in the media and permit the growth of typhi and paratyphi organism. These mechanism works as the typhoid organism growth, sodium sulphate present in the media react with Bismuth ammonium citrate to form bismuth sulphate. The present of bismuth sulphate and glucose in the media yield black colonies. These only take place if the media is not too acidic, in case of too much acid in the media, sodium phosphate can be added to inhibit the acidic content of the media. Ferric citrate can also be added to inhibit the toxicity of the bismuth substance present in the media. Salmonellae typhi colonies show black color when left for at least 24 hours. While the salmonellae paratyphi show black color of colonies when left at least 48 hours. In case of coliform organism it also inhibited by brilliant green, also sodium sulphate can be presented by bismuth sulphate in the media.

Selenite F media: the media is a modified formed.

Composition

Sodium acid selenite

Manitol

Peptone

Disodium hydrogen orthophosphate

Preparation follows manufacturer instruction.

The sodium acid selenite in the media has a pH value closer to 7.0 which is the neutral point, and this pH value is very poisonous to E. coli, but harmless to salmonellae micro organism, as the increase in the pH value of the media so the decrease poisonous content of the media, but in other to bring the pH of these media to normal value buffer can be used to normalized the media pH concentration. In the case of media too alkaline fermentation of carbohydrate can be introduced to produce acid and reduce the alkaline content of the media. So reduce the alkaline concentration produce when bacteria brake down sodium acid selenite is very important to be used in these media, but care should be taken because the chemical is poisonous when invalid.

Phenylalanine Agar

Composition

Yeast extract

D-phenylalanine

Sodium chloride

Agar

Disodium hydrogen orthophosphate

Preparation follows manufacturer instruction

Phenylalanine is converted to phenylpyruvic acid by action of enzyme called phenylalanase, these process reduce the amino content of these substance and form exact property of proteous and prepared a better way for the feature of the organism. The phenylpyruvic acid can be confirmed in these media by addition of ion chloride which will result in the green coloration of the present of phenylpyruvic acid in the media.

ISOLATION OF CORYNECBACTERIA

Several media used for isolation of corynecbacterium diphthereae are from mixed culture that is made up of tellurium constituent. The tellurium substance in these media prevents the growth of any other micro organism as soon as this micro organism is isolated from the mixed, it be follow by subculture. This culture can be control in a serum media.

Hoyles Media: these media are used for mixture culture of corynecbacteria diphthereae.

Composition

Beef extract

Sodium chloride

Proptease

Peptone

Agar

The composed will first heated and melted to liquid before laked horse blood and potassium tellurite solution is added.

laked horse blood: to achieved these substance, blood may be freezing and make soft several times, or 0.5ml of 10% white saponin can be added, after it should be sterilized with autoclave. After the completion of sterilization which will take place for 24-48 hours, then 10ml of the blood and inoculated with the sample in aqueous and incubated. Colonies of gavis type show a state-gray color with bluish tinage. The shape of thecolonies in the culturevis of that of daisy head, size and color of these colonies is that similar to that of gavis type, but appear more shining, convex and perfectively circle outline. The colonies having a fiddle place type and never bigger than 2mm darker than other colonies and have a shape of that of cook egg in water without shell. These method described above are the old method, to have it own way of description.

Loeffers serum: these usually a slope culture media which they used for pure culture of corynecbacterium diphthereae. The media is made up of 3ml ox serum and 2% of 1ml of glucose broth. Method of preparation are, the glucose is sterilized at 115ºC for 15 minute in an autoclave, serum should be sterilized by filtering with the used of seitz filtration techniques. Mixed the substance and aseptically distributed to the tube or bottle, heat the media at 75 ºC until it set, heat at 75 ºC for I hour in the next day, but care should be taken that the temperature did not exceed 75 ºC.

Egg media: these the media prepared for the purpose of cultivation of tubercle bacillus, the media can be purchase commercially in the market.

Another media prepared for the cultivation of mycobacterium is Lowenstein-Jensen media, the media is very important for the isolation of mycobacterium tuberculosis. The media is made up of having malathite as an inhibitory agent that inhibit the growth of other in the culture plate some introduction of antibiotic is initiated in the media in other to prevent some contaminant organism that may growth in the culture plate.

Glycerol Egg: these media specifically made for culturing of human tubercle bacillus, this micro organism growth well in a media that is incorporated with glycerol.

Composition

Egg 78%

Nutrient broth 20%

Glycerol 2%

Preparation

Nutrient is sterilized at a temperature of 115 ºC for 15minute in an autoclave, rough egg is brake in an aseptic condition into a sterile flask incorporated with a glass beads. Before the nutrient agar glycerol is added, to the media, shake the flask with egg inside filter via muslin into the glycerol broth. Distribute into bottle or tube via aseptic condition and inspissate in a slopping position at 75-80 ºC heat the media until the solidification take place.

Dorsal Egg: this media composed of

Nutrient broth dissolved

Saline 20ml

Egg 80ml

Preparation, the same as glycerol preparation, the media is specifically made to culture human and bovine tubercle bacilli.

Lowesten-Jensen media: this media is specifically produce for the cultivation of human tubercle bacilli, but can also be used to growth bovine tubercle bacilli, these can take place by the addition of 0.5% of sodium pyruvate. The media is made is made up of asparagines mineral salt solution

.

Composition

Glycerol 20%

Asparagines 0.6%

Magnesium citrate 0.1%

Magnesium 0.04%

Potassium dihydrogen orthophosphate 0.4%

After the substance is combined together, it should be steam for 2 hours and store in a refrigerator, your asparagines solution has successfully been produce.

Now the step on how to produce the media are; measure the amount of volume of the asparagines mineral salt solution required to produce the media, for example of you measure 150ml of asparagines mineral salt, then 6g of potato flour to the solution heat the mixture with constant stirring until homogenous solution is achieved. Then autoclave the mixture for 115ºC for 20 minute, break 5 egg under aseptic condition into a sterile flask, the flask should contained glass bead, shake the eggs in the flask, after that filter with a sterile muslin. Combine the egg with asparagines potato ach mixed add 2% of 5ml of malachite green solution, finally distribute under aseptic condition in a bottle or tube and sterile by inspissating at 75ºC to 80ºC until solidification of the media is achieved.

Asparagines is a mineral source for tubercle bacilli, and glycerol act as a carbon source for the micro organism, while malachite green is an inhibitory agent that inhibit the growth of other micro organism in the media. Examination of human tubercle bacilli show yellow colonies of dry heap up, while that of bovine show small degree grow of colorless colonies.

Media for isolation of staphylococci: there are two form media for the isolation they are; baired packed agar and manitol media.

Baired packed Agar: this is a selective media used for cultivation of staphylococci micro organism. The media work in a coagulate process of the positive staphylococci.

Composition

Tryptone

Beef extract

Glycine

Agar

Egg York

Potassium tellurite

Lithium chloride

Sodium pyruvate

The potassium tellurite added to the media is an inhibitory agent that inhibits the growth of other micro organism in the plate, and pyruvate and glycine is a stimulant agent it stimulate the growth of staphylococci. The organism growth as a black shiny convex colonies by the surrounding a clear surface area, while coagulase negative organism do not possess the feature. The phenomenon occur doe the present of tellurite and egg York in the media, this substance aid in identification and differentiation of the organism.

Preparation is base on the manufacturer instruction.

Manitol salt sugar: this media also use for examination of staphylococci organism.

Composition

Peptone

Sodium

Ager

Manitol

Phenol red

The media is often used for research purpose.

Preparation based on the manufacturer instruction

Phenol red act as an indicator in the media, for identification of coagulase positive colonies, on this identification, coagulase negative staphylococci is surrounded in a reddish color, of the phenol red, while coagulase positive staphylococci colonies surrounded in a bright orange yellow surface area.

Before coagulase test are carried out, subculture of the organism should be carried out in a media that does not contain much salt.

Cultivation of vibrio cholerae: this micro organism is culture in a thiosulphate citrate bile salt (TCBS) these organism can be identify through a process of fermentation of sucrose in the media.

Composition

Agar

Bromothymol blue

Ferric citrate

Peptone

Sodium citrate

Yearst extract

Sodium thiosulphate

Sodium taurocholate

The colonies of the organism appear yellow on the bluish green background of the medium after 10-18 hours incubation of the culture plate.

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