culture media in oral pathology

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Culture Media in Oral Pathology

Pardeep Goyal*, Deepak Goyal**, Ashish Aggarwal***, Imtiyaz Nadaf****,Indu Goyal*****.

-5-Journal of Dental Esthetic and Function- Vol 2, Issue 1, Jan-June 2013.

Review Article

INTRODUCTION:

diagnostic bacteriology laboratory

have their origins, for the most part,

back in the 'Golden age of bacteriology'

in the late nineteenth and twentieth

centuries. The objectives of early

medium designed were to growpathogenic bacteria, separate them

from the other organisms present in

samples and ultimately, differentiate

their phenotypical properties so that

they could be identified. A critical

development was introduction of1

solidifying reagents.

Ideal culture media characteristics

Culture media gives artificial

environment simulating natural

conditions necessary for growth ofbacteria.

1. Must give satisfactory growth

from single inoculums.

2. Should give rapid growth.

3. Should be easy to grow.

4. Should be reasonably cheap.

5. Should be easily reproducible.

The media used in medical

6. Should enable to demonstrateall characteristics in which weare interested.

HISTORY

The earliest culture media werel iquid, which made the

isolation of bacteria to preparep ur e c u l t u r e e x t re mel ydifficult.

The original media used byLOUIS PASTEUR used liquidssuch as urine or meat broth.

In 1881, ROBERT KOCHpublished an article describingthe use of boiled potatoes slicedwith a flame sterilized knife, inculturing media.

A t a b o u t s a m e t i m eFREDERICK LOEFFLER, anassociate of Koch's, developeda meat extract peptone mediumfor cultivating pathogenicbacteria. Koch then tried tosolidify this medium by usinggelatin. The new solid mediumworked well but it would not be

ABSTRACT :

One of the most important reasons for culturing bacteria in vitro is its utility indiagnosing infectious diseases. Isolating a bacterium from sites in body normallyknown to be sterile is an indication of its role in the disease process. Culturing bacteriais also the initial step in studying its morphology and its identification. Bacteria have tobe cultured in order to obtain antigens from developing serological assays or vaccinesCertain genetic studies and manipulations of the cells also need that bacteria becultured in vitro. Culturing bacteria also provide a reliable way estimating theirnumbers. Culturing on solid media is another convenient way of separating bacteria inmixtures.

Keywords:Bacteria, Infection, Growth

Address forCorrespondance

Dr. Pardeep KumarS/O Sh. Jagdish Rai

H No.13243-B, Street-11Namdev Marg

Bathinda -151001Punjab

India

*Senior Lecturer, Departmentof Oral and Maxillofacial

Pathology, Adesh Institute ofDental Sciences & research,

Bathinda (Punjab)**Senior Lecturer, Department

of Oral and MaxillofacialPathology, Jan Nayak

Ch. Devi Lal Dental College,Sirsa (Haryana)

***Senior Lecturer,Department of Oral Medicine

& Radiology, Institute ofDental Sciences, Bareilly

(Uttar Pradesh)****Reader, Department of

Oral and MaxillofacialPathology, Adesh Institute of

Dental Sciences & Research,Bathinda (Punjab)

*****Tutor, Department of Oraland Maxillofacial Pathology,

Adesh Institute of DentalSciences & Research,

Bathinda (Punjab)


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incubated at 37C (the best temperature forhuman bacterial pathogens) because thegelatin would melt. Furthermore, somebacteria digested gelatin.

BASIC REQUIREMENTS OF CULTURE MEDIA

1. Energy source

2. Carbon source

3. Nitrogen source

4. Salts like sulfates, phosphates, chlorides andcarbonates of sodium, potass ium,magnesium, ferric, calcium and traceelements like copper etc.

5. Satisfactory pH 7.2-7.6

6. Adequate oxidation reduction potential.

7. Growth factors like tryptophan for Salmonellatyphi.

COMMON INGREDIENTS OF CULTURE

MEDIA

Water, agar, peptone, casein hydrolysate, meatextract, yeast extract, blood, serum.

WATER

Tap water is often suitable for culture media,particularly, if it has a low mineral content, but if thelocal supply is found unsuitable, glass-distilled ordemineralised water must be used instead. Small

amounts of copper are highly inhibitory to bacterialgrowth so that copper-distilled water cannot be usedfor media. Suitable demineralisers are commerciallyavailable.

AGAR

Agar-agar, or 'agar' for short, is prepared froma clarified, dried material and supplied as a powder.There are considerable differences in the properties ofthe agars manufactured in different places, and evenbetween different batches from the same source. Inwatery solutions, agar gives a firm gel that remainsunmelted at all incubation temperatures and it isgenerally bacteriologically inert, being decomposedor liquefied by only a few varieties of bacteria. Inthese respects, it is more suitable than gelatin; a 15%solution of gelatin melts at 24C and gelatin isdecomposed by many proteolytic bacteria. Agar doesnot add to the nutritive properties of a medium and asuitable agar should be free from growth-promoting

as well as growth-inhibiting substances. The exactconcentration to be used may require someadjustment according to the other constituents of themedium. A concentration of 1-2% usually yields asuitable gel, but the manufacturer's instructionsshould be followed.

PEPTONE

Peptone consists of water soluble productsobtained from lean meat or other protein materiasuch as heart muscle, casein, fibrin or soya flourusually by digestion with the proteolytic enzymespeps in, trypsin and papain. The importantconstituents are peptones, proteases, amino acids, avariety of inorganic salts including phosphatespotassium and magnesium, and certain accessorygrowth factors such as nicotinic acid and riboflavin.

Peptone is supplied as a golden granular

powder with a low moisture content, preferably under5% and usually a slight acid reaction, gives a pHbetween 5 and 7 in a 1% solution. It is hygroscopicand soon becomes sticky when exposed to air; stockbottles shuld therefore be kept firmly closed.

CASEIN HYDROLYSATE

This consists largely of the amino acidsobtained by hydrolysis of the milk protein casein. Italso contains phosphate and other salts, and certain

growth factors. Hydrolysis is effected either withhydrochloric acid or with a proteolytic enzyme.

The acid hydrolysate is poorer nutritionallybecause tryptophan is largely destroyed during thehydrolysis and some other amino acids are reduced inamount, tryptophan must therefore be added to themedium to make it suitable for tryptophan requiringbacteria.

MEAT EXTRACT

Commercially prepared meat extract is used

as a substitute for an infusion of fresh meat. A typicacommercial preparation contains a wide variety ofwater soluble compounds, including proteindegradation products, e.g. gelatin, albuminosespeptones, proteoses and amino acids and other aminocompounds such as creatine, creatinine, carnosineanserine, purine and glutathione; it also containsmany mineral salts (KH PO and NaCl most2 4abundantly), accessory growth factors (e.g. thiamine


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nicotinic acid, riboflavin, pyridoxine, pantothenicacid and choline) and some carbohydrates.

YEAST EXTRACT

Commercial yeast extract is prepared fromwashed cells of Brewer's or Baker's yeast. It containsa wide range of amino acids (amounting to nearly

50% of its mass) growth factors (especially ofVitamin B group) and inorganic salts (particularlypotassium and phosphate). Over 10% of yeast extractis carbohydrate including glycogen, trehalose andpentoses. Yeast extract is used mainly as acomprehensive source of growth factors and may besubstituted for meat extract in culture media.

BLOOD

Blood for use in the media must be collected

with aseptic precautions adequate to exclude bacterialcontamination and preserve the blood in its originalsterile condition. It must be rendered non-coagulatingby defibrination or by the addition of citrate oroxalate; defibrination is recommended because itinvolves no additive that might alter the nutritiveproperties of the medium.

Sterile horse blood can be preparedcommercially. Alternatively, blood may be collectedfrom rabbits and other laboratory animals, sheep,oxen and horses at the abattoir or from man.

SERUM

Serum for use in media need not be collectedwith aseptic precautions because it can be filter-sterilized. Sterile animal sera can be obtainedcommercially.

Serum may also be prepared from unsteriledefibrinated or oxalated blood. Serum is sterilized byfilteration through a suitable membrane filter or Seitzfilter. It may be stored at 3-5C in the refrigerator untilrequired for use.

CONTAINERS FOR MEDIA AND CULTURE

Glass vessels stoppered with cotton wool,test-tubes stoppered with cotton wool or with slip onmetal caps and screw capped bottles of differentcapacities and shapes can be used as containers forliquid media and culture.

Petri dishes are shallow flat-bottomedcircular clear glass or plastic containers with lids;

they are normally 90mm in diameter, but smaller andlarger dishes are available for special purposes.

STERILIZATION OF CULTURE MEDIA

The choice of method to be used to sterilize amedium depends on whether or not the ingredients aredecomposed by heat. If autoclaving will not damage

the medium, it is the best method of sterilization. Iautoclaving will not damage the medium, it is the bes

2method of sterilization.

If any of the ingredients of a medium are liableto be spoiled by autoclaving, the complete mediumshould be sterilized by heat. In such cases, it is usual toautoclave the thermostable ingredients of the mediumand to add the sterile heat sensitive ingredients withsterile precautions. Some sensitive ingredients likeblood, serum or egg-yolk can be obtained sterile fromcommercial sources. Others must be sterilized by

3filtration through a bacterial filter.Some selective media that cannot be

autoclaved contain ingredients that are inhibitory tomost probable contaminants. These media aresometimes prepared without the sterilization of someingredients, reliance being placed on the inhibitors tosuppress contaminants. The method is usuallysuccessful but must always be regarded as less thanideal.

TYPES OF CULTURE MEDIA

Culture media can be classified into followingtypes

I. On the basis of 'Physical State'

a. Liquid

b. Semi-solid

c. Solid

II. On the basis of presence of molecular oxygenand reducing substances in the media

a. Aerobic

b. Anaerobic

III. On the basis of nutritional factors

a. Simple

b. Complex

c. Synthetic/defined

d. Special enriched, enrichmentselective indicator/differential, sugartransport


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SIMPLE MEDIA / BASIC MEDIA

Nutrient broth is an example of simplemedium. It consists of peptone, meat extract,sodium chloride and water.

Nutrient agar, made by adding 2% agar tonutrient broth is the simplest and mostcommon medium in routine diagnostic

laboratories. If the concentration of agar isreduced to 0.2 0.5%, semisolid or sloppyagar is obtained which enables motileorganisms to spread. Increasing theconcentration of agar to 6% preventsspreading or swarming by organisms such asProteus.

Basal media such as nutrient broth andpeptone water are generally used as simplemedia and as the basis of supplemented

4.5\enriched media.

There are 3 types of nutrient broth:

1. Meat infusion broth

2. Meats extract broth

3. Digest broth

SYNTHETIC / DEFINED MEDIA

Some micro organisms, particularlyphotoli thotropic autotro phs such ascyanobacteria and eukaryotic algae, can begrown on relatively simple media containing

CO as carbon source (often added as sodium2carbonate and bicarbonate), nitrate orammonia as a nitrogen source, sulfate,phosphate and a variety of minerals. Such amedia in which all components are known as'Defined medium' or 'Synthetic medium'.

Simple synthetic media

Complex synthetic media

SELECTIVE MEDIA

Selective media contains substances thatinhibit all but a few types of bacteria. They facilitateisolation of particular species from a mixedinoculum. Selective media are solid in contrast toenrichment media which are liquid.

Examples:

1. Deoxycholate agar (DCA): addition ofdeoxycholate acts as a selective agent forenteric bacilli (Salmonella, Shigella).

2. Bile Salt Agar (BSA): bile salt is a selectiveagent which favours the growth of only Vibriocholerae whereas inhibits the growth of otherintestinal micro-organisms.

3. Wilson's and Blair medium for Salmonella.

4. L o w e n s t e i n - J e n s e n m e d i u m f oMycobacterium tuberculosis.

ENRICHED MEDIA

When nutrients such as blood, serum or eggare added to basal medium, it is called ENRICHEDMEDIUM. These media are employed to groworganisms which are more exacting in their nutritionaneeds.

Examples:

1. Blood agar: blood is added to nutrient agarused for isolation of Staphylococcus.

2. Chocolate agar: for isolation of Hemophiluand Neisseria.

3. Bordet-Gengou media: for isolation oBordetella.

4. Loeffler's serum slope: for isolation oCorynebacterium diphtheria.

ENRICHMENT MEDIA

In mixed cultures or in material containingmore than one bacterium, the bacterium to be isolated

is often overgrown by the unwanted bacteria. Usuallythe nonpathogenic or commensal bacteria tend toovergrow the pathogenic ones, e.g. Salmonella typhbeing overgrown byE.coliin cultures from feces. Insuch a situation substances which have a stimulatoryeffect on the bacteria to be grown or inhibitory effecon those to be suppressed are incorporated in themedium. If such substances are added to a liquidmedium, the result is an absolute increase in thenumbers of wanted bacterium relative to otherbacteria. Thus, when substances which have

stimulatory effect on bacteria to be grown orinhibitory effect on the undesired ones are added to aliquid medium, it is called as ENRICHMENT

7MEDIA.

Examples:

Selenite F broth

Tetrathione broth: where tetrathionate inhibitscoliforms while allowing typhoid-paratyphoid bacillto grow freely.


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DIFFERENTIAL / INDICATOR MEDIA

When a substance is added into a mediumwhich would produce a visible change in themedium following the growth of particularorganism, it is designated as Indicator Media.

Examples:

1. Incorporation of sulphite in Wilson and

Blair's medium. S.typhi reduces sulphite andsulphide in the presence of glucose and thecolonies of S.typhi have a metallic blacksheen.

2. Potassium tellurite in McLeod's medium getsreduced to metallic tellurium by theDiphtheria bacillus to produce blackcolonies.

3. MacConkey agar is a useful medium forcultivation of enterobacteria. It contains a bilesalt t inhibit non-intestinal bacteria and

lactose with neutral red to distinguish Lactosefermenting (LF) coliforms from non-lactosefermenting Salmonella and Shigella species.The concentration of sodium taurocholatemay be reduced to suit less tolerant

8organisms.

4. Blood agar distinguishes between hemolyticand non-hemolytic bacteria. Hemolyticb a c t e r i a ( m a n y s t r e p t o c o c c i a n dstaphylococci isolated from throat) produceclear zones around their colonies because of

red cells destruction.

SUGAR MEDIA

The term sugar in microbiology denotes anyfermentable substance. Glucose, lactose, sucrose andmannitol are routinely employed for fermentationtests.

The usual sugar media consists of the sugar inpeptone water alongwith an appropriate indicator(Andrade's indicator 0.005% acid fuchsin in 1NNaOH). A small tube (durham's tube) is kept inverted

in the larger sugar tube to detect gas production. Thecolorless medium turns pink with production of acidby bacteria and gas production is indicated by gasbubbles accumulated in Durham's tube.

SUGAR FERMENTATION MEDIUM:

Composition:

Peptone 15g

Andrade's Indicator 10ml

Sugar to be tested 20g

Water 1 L

Dissolve the peptone and Andraed's indicatorin 1 litre of water and 20g of sugar. Distribute 3mlamounts in standard test tubes containing an invertedDurham's tube. Sterilize by steaming at 100C for

30min for 3 consecutive days.

TRANSPORT MEDIA

In case of delicate organisms (e.g. Gonococciwhich do not survive the time taken for transportingthe specimen to the laboratory or may be overgrownby non-pathogens (e.g. dysentery or choleraorganisms), special media are devised and these aretermed 'Transport Media'. E.g. Stuart's medium anon-nutrient soft agar gel containing a reducing agento prevent oxidation, and charcoal to neutralize

certain bacterial inhibitors for gonococci, and9

buffered glycerol saline for enteric bacilli.

ANAEROBIC MEDIA

These media are used to grow anaerobicmicroorganisms. Example: Robertson's cooked meamedium for Clostridium tetani.

MEDIA FOR BIOCHEMICAL TESTS

Triple Sugar Iron agar test for hydrogen10sulphide production.

Catalase test

Oxidase test

Urease test

RECENT ADVANCES IN CULTURE MEDIA

Candida albicanswild type or mutanstrain interaction with epithelial tissue or theevaluation of the host immune response usinghistological, biochemical and molecular methods. A

such, the models can be utilized as a tool to investigatecellular interactions or protein and gene expressionthat are not complicated by non-epithelial factors. Tostudy the impact of innate immunity or the antifungaactivity of natural and non-natural compounds, themucosal infection models can be supplemented withimmune cells, antimicrobial agents or probioticbacteria. The model requires at least 3 days to beestablished and can be maintained thereafter for 24days.


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culture media in oral pathology

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