Review Article Culture Media in Oral Pathology

Pardeep Goyal*, Deepak Goyal**, Ashish Aggarwal***, Imtiyaz Nadaf****, Indu Goyal*****.
*Senior Lecturer, Department of Oral and Maxillofacial Pathology, Adesh Institute of Dental Sciences & research, Bathinda (Punjab) **Senior Lecturer, Department of Oral and Maxillofacial Pathology, Jan Nayak Ch. Devi Lal Dental College, Sirsa (Haryana) ***Senior Lecturer, Department of Oral Medicine & Radiology, Institute of Dental Sciences, Bareilly (Uttar Pradesh) ****Reader, Department of Oral and Maxillofacial Pathology, Adesh Institute of Dental Sciences & Research, Bathinda (Punjab) *****Tutor, Department of Oral and Maxillofacial Pathology, Adesh Institute of Dental Sciences & Research, Bathinda (Punjab)

ABSTRACT : One of the most important reasons for culturing bacteria in vitro is its utility in diagnosing infectious diseases. Isolating a bacterium from sites in body normally known to be sterile is an indication of its role in the disease process. Culturing bacteria is also the initial step in studying its morphology and its identification. Bacteria have to be cultured in order to obtain antigens from developing serological assays or vaccines. Certain genetic studies and manipulations of the cells also need that bacteria be cultured in vitro. Culturing bacteria also provide a reliable way estimating their numbers. Culturing on solid media is another convenient way of separating bacteria in mixtures. Keywords: Bacteria, Infection, Growth INTRODUCTION: The media used in medical 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 grow pathogenic 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 of 1 solidifying reagents. Ideal culture media characteristics Culture media gives artificial environment simulating natural conditions necessary for growth of bacteria. 1. 2. 3. 4. 5. Must give satisfactory growth from single inoculums. Should give rapid growth. Should be easy to grow. Should be reasonably cheap. Should be easily reproducible.
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Should enable to demonstrate all characteristics in which we are interested.

Address for Correspondance Dr. Pardeep Kumar S/O Sh. Jagdish Rai H No.13243-B, Street-11 Namdev Marg Bathinda -151001 Punjab India

HISTORY The earliest culture media were liquid, which made the isolation of bacteria to prepare pure culture extremely difficult. The original media used by LOUIS PASTEUR used liquids such as urine or meat broth. In 1881, ROBERT KOCH published an article describing the use of boiled potatoes sliced with a flame sterilized knife, in culturing media. At about same time, FREDERICK LOEFFLER, an associate of Koch's, developed a meat extract peptone medium for cultivating pathogenic bacteria. Koch then tried to solidify this medium by using gelatin. The new solid medium worked well but it would not be

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incubated at 37C (the best temperature for human bacterial pathogens) because the gelatin would melt. Furthermore, some bacteria digested gelatin. BASIC REQUIREMENTS OF CULTURE MEDIA 1. Energy source 2. Carbon source 3. Nitrogen source 4. Salts like sulfates, phosphates, chlorides and carbonates of sodium, potassium, magnesium, ferric, calcium and trace elements like copper etc. 5. Satisfactory pH 7.2-7.6 6. Adequate oxidation reduction potential. 7. Growth factors like tryptophan for Salmonella typhi. COMMON INGREDIENTS OF CULTURE MEDIA Water, agar, peptone, casein hydrolysate, meat extract, yeast extract, blood, serum. WATER Tap water is often suitable for culture media, particularly, if it has a low mineral content, but if the local supply is found unsuitable, glass-distilled or demineralised water must be used instead. Small amounts of copper are highly inhibitory to bacterial growth so that copper-distilled water cannot be used for media. Suitable demineralisers are commercially available. AGAR Agar-agar, or 'agar' for short, is prepared from a clarified, dried material and supplied as a powder. There are considerable differences in the properties of the agars manufactured in different places, and even between different batches from the same source. In watery solutions, agar gives a firm gel that remains unmelted at all incubation temperatures and it is generally bacteriologically inert, being decomposed or liquefied by only a few varieties of bacteria. In these respects, it is more suitable than gelatin; a 15% solution of gelatin melts at 24C and gelatin is decomposed by many proteolytic bacteria. Agar does not add to the nutritive properties of a medium and a suitable agar should be free from growth-promoting
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as well as growth-inhibiting substances. The exact concentration to be used may require some adjustment according to the other constituents of the medium. A concentration of 1-2% usually yields a suitable gel, but the manufacturer's instructions should be followed. PEPTONE Peptone consists of water soluble products obtained from lean meat or other protein material such as heart muscle, casein, fibrin or soya flour, usually by digestion with the proteolytic enzymes pepsin, trypsin and papain. The important constituents are peptones, proteases, amino acids, a variety of inorganic salts including phosphates, potassium and magnesium, and certain accessory growth factors such as nicotinic acid and riboflavin. Peptone is supplied as a golden granular powder with a low moisture content, preferably under 5% and usually a slight acid reaction, gives a pH between 5 and 7 in a 1% solution. It is hygroscopic and soon becomes sticky when exposed to air; stock bottles shuld therefore be kept firmly closed. CASEIN HYDROLYSATE This consists largely of the amino acids obtained by hydrolysis of the milk protein casein. It also contains phosphate and other salts, and certain growth factors. Hydrolysis is effected either with hydrochloric acid or with a proteolytic enzyme. The acid hydrolysate is poorer nutritionally because tryptophan is largely destroyed during the hydrolysis and some other amino acids are reduced in amount, tryptophan must therefore be added to the medium to make it suitable for tryptophan requiring bacteria. MEAT EXTRACT Commercially prepared meat extract is used as a substitute for an infusion of fresh meat. A typical commercial preparation contains a wide variety of water soluble compounds, including protein degradation products, e.g. gelatin, albuminoses, peptones, proteoses and amino acids and other amino compounds such as creatine, creatinine, carnosine, anserine, purine and glutathione; it also contains many mineral salts (KH2PO4 and NaCl most abundantly), accessory growth factors (e.g. thiamine,
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nicotinic acid, riboflavin, pyridoxine, pantothenic acid and choline) and some carbohydrates. YEAST EXTRACT Commercial yeast extract is prepared from washed cells of Brewer's or Baker's yeast. It contains a wide range of amino acids (amounting to nearly 50% of its mass) growth factors (especially of Vitamin B group) and inorganic salts (particularly potassium and phosphate). Over 10% of yeast extract is carbohydrate including glycogen, trehalose and pentoses. Yeast extract is used mainly as a comprehensive source of growth factors and may be substituted for meat extract in culture media. BLOOD Blood for use in the media must be collected with aseptic precautions adequate to exclude bacterial contamination and preserve the blood in its original sterile condition. It must be rendered non-coagulating by defibrination or by the addition of citrate or oxalate; defibrination is recommended because it involves no additive that might alter the nutritive properties of the medium. Sterile horse blood can be prepared commercially. Alternatively, blood may be collected from rabbits and other laboratory animals, sheep, oxen and horses at the abattoir or from man. SERUM Serum for use in media need not be collected with aseptic precautions because it can be filtersterilized. Sterile animal sera can be obtained commercially. Serum may also be prepared from unsterile defibrinated or oxalated blood. Serum is sterilized by filteration through a suitable membrane filter or Seitz filter. It may be stored at 3-5C in the refrigerator until required for use. CONTAINERS FOR MEDIA AND CULTURE Glass vessels stoppered with cotton wool, test-tubes stoppered with cotton wool or with slip on metal caps and screw capped bottles of different capacities and shapes can be used as containers for liquid media and culture. Petri dishes are shallow flat-bottomed circular clear glass or plastic containers with lids;
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they are normally 90mm in diameter, but smaller and larger dishes are available for special purposes. STERILIZATION OF CULTURE MEDIA The choice of method to be used to sterilize a medium depends on whether or not the ingredients are decomposed by heat. If autoclaving will not damage the medium, it is the best method of sterilization. If autoclaving will not damage the medium, it is the best method of sterilization.2 If any of the ingredients of a medium are liable to be spoiled by autoclaving, the complete medium should be sterilized by heat. In such cases, it is usual to autoclave the thermostable ingredients of the medium and to add the sterile heat sensitive ingredients with sterile precautions. Some sensitive ingredients like blood, serum or egg-yolk can be obtained sterile from commercial sources. Others must be sterilized by filtration through a bacterial filter.3 Some selective media that cannot be autoclaved contain ingredients that are inhibitory to most probable contaminants. These media are sometimes prepared without the sterilization of some ingredients, reliance being placed on the inhibitors to suppress contaminants. The method is usually successful but must always be regarded as less than ideal. TYPES OF CULTURE MEDIA Culture media can be classified into following types I. On the basis of 'Physical State' a. Liquid b. Semi-solid c. Solid II. On the basis of presence of molecular oxygen and reducing substances in the media a. Aerobic b. Anaerobic On the basis of nutritional factors a. Simple b. Complex c. Synthetic/defined d. Special enriched, enrichment, selective indicator/differential, sugar, transport

III.

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SIMPLE MEDIA / BASIC MEDIA Nutrient broth is an example of simple medium. It consists of peptone, meat extract, sodium chloride and water. Nutrient agar, made by adding 2% agar to nutrient broth is the simplest and most common medium in routine diagnostic laboratories. If the concentration of agar is reduced to 0.2 0.5%, semisolid or sloppy agar is obtained which enables motile organisms to spread. Increasing the concentration of agar to 6% prevents spreading or swarming by organisms such as Proteus. Basal media such as nutrient broth and peptone water are generally used as simple media and as the basis of supplemented enriched media.4.5\ There are 3 types of nutrient broth: 1. Meat infusion broth 2. Meats extract broth 3. Digest broth SYNTHETIC / DEFINED MEDIA Some micro organisms, particularly photolithotropic autotrophs such as cyanobacteria and eukaryotic algae, can be grown on relatively simple media containing CO2 as carbon source (often added as sodium carbonate and bicarbonate), nitrate or ammonia as a nitrogen source, sulfate, phosphate and a variety of minerals. Such a media in which all components are known as 'Defined medium' or 'Synthetic medium'. Simple synthetic media Complex synthetic media SELECTIVE MEDIA Selective media contains substances that inhibit all but a few types of bacteria. They facilitate isolation of particular species from a mixed inoculum. Selective media are solid in contrast to enrichment media which are liquid. Examples: 1. Deoxycholate agar (DCA): addition of deoxycholate acts as a selective agent for enteric bacilli (Salmonella, Shigella).
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2.

3. 4.

Bile Salt Agar (BSA): bile salt is a selective agent which favours the growth of only Vibrio cholerae whereas inhibits the growth of other intestinal micro-organisms. Wilson's and Blair medium for Salmonella. Lowenstein-Jensen medium for Mycobacterium tuberculosis.

ENRICHED MEDIA When nutrients such as blood, serum or egg are added to basal medium, it is called ENRICHED MEDIUM. These media are employed to grow organisms which are more exacting in their nutritional needs. Examples: 1. Blood agar: blood is added to nutrient agar, used for isolation of Staphylococcus. 2. Chocolate agar: for isolation of Hemophilu and Neisseria. 3. Bordet-Gengou media: for isolation of Bordetella. 4. Loeffler's serum slope: for isolation of Corynebacterium diphtheria. ENRICHMENT MEDIA In mixed cultures or in material containing more than one bacterium, the bacterium to be isolated is often overgrown by the unwanted bacteria. Usually the nonpathogenic or commensal bacteria tend to overgrow the pathogenic ones, e.g. Salmonella typhi being overgrown by E.coli in cultures from feces. In such a situation substances which have a stimulatory effect on the bacteria to be grown or inhibitory effect on those to be suppressed are incorporated in the medium. If such substances are added to a liquid medium, the result is an absolute increase in the numbers of wanted bacterium relative to other bacteria. Thus, when substances which have stimulatory effect on bacteria to be grown or inhibitory effect on the undesired ones are added to a liquid medium, it is called as ENRICHMENT MEDIA.7 Examples: Selenite F broth Tetrathione broth: where tetrathionate inhibits coliforms while allowing typhoid-paratyphoid bacilli to grow freely.
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DIFFERENTIAL / INDICATOR MEDIA When a substance is added into a medium which would produce a visible change in the medium following the growth of particular organism, it is designated as Indicator Media. Examples: 1. Incorporation of sulphite in Wilson and Blair's medium. S.typhi reduces sulphite and sulphide in the presence of glucose and the colonies of S.typhi have a metallic black sheen. 2. Potassium tellurite in McLeod's medium gets reduced to metallic tellurium by the Diphtheria bacillus to produce black colonies. 3. MacConkey agar is a useful medium for cultivation of enterobacteria. It contains a bile salt t inhibit non-intestinal bacteria and lactose with neutral red to distinguish Lactose fermenting (LF) coliforms from non-lactose fermenting Salmonella and Shigella species. The concentration of sodium taurocholate may be reduced to suit less tolerant 8 organisms. 4. Blood agar distinguishes between hemolytic and non-hemolytic bacteria. Hemolytic bacteria (many streptococci and staphylococci isolated from throat) produce clear zones around their colonies because of red cells destruction. SUGAR MEDIA The term sugar in microbiology denotes any fermentable substance. Glucose, lactose, sucrose and mannitol are routinely employed for fermentation tests. The usual sugar media consists of the sugar in peptone water alongwith an appropriate indicator (Andrade's indicator 0.005% acid fuchsin in 1N NaOH). A small tube (durham's tube) is kept inverted in the larger sugar tube to detect gas production. The colorless medium turns pink with production of acid by bacteria and gas production is indicated by gas bubbles accumulated in Durham's tube. SUGAR FERMENTATION MEDIUM: Composition: Peptone 15g Andrade's Indicator 10ml
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Sugar to be tested Water

20g 1L

Dissolve the peptone and Andraed's indicator in 1 litre of water and 20g of sugar. Distribute 3ml amounts in standard test tubes containing an inverted Durham's tube. Sterilize by steaming at 100C for 30min for 3 consecutive days. TRANSPORT MEDIA In case of delicate organisms (e.g. Gonococci) which do not survive the time taken for transporting the specimen to the laboratory or may be overgrown by non-pathogens (e.g. dysentery or cholera organisms), special media are devised and these are termed 'Transport Media'. E.g. Stuart's medium a non-nutrient soft agar gel containing a reducing agent to prevent oxidation, and charcoal to neutralize certain bacterial inhibitors for gonococci, and buffered glycerol saline for enteric bacilli.9 ANAEROBIC MEDIA These media are used to grow anaerobic microorganisms. Example: Robertson's cooked meat medium for Clostridium tetani. MEDIA FOR BIOCHEMICAL TESTS Triple Sugar Iron agar test for hydrogen sulphide production.10 Catalase test Oxidase test Urease test RECENT ADVANCES IN CULTURE MEDIA Candida albicans wild type or mutant strain interaction with epithelial tissue or the evaluation of the host immune response using histological, biochemical and molecular methods. As such, the models can be utilized as a tool to investigate cellular interactions or protein and gene expression that are not complicated by non-epithelial factors. To study the impact of innate immunity or the antifungal activity of natural and non-natural compounds, the mucosal infection models can be supplemented with immune cells, antimicrobial agents or probiotic bacteria. The model requires at least 3 days to be established and can be maintained thereafter for 24 days.
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Candida-produced farnesol can induce apoptosis in vitro in oral squamous cell carcinoma (OSCC) lines. Cell proliferation, apoptosis, mitochondrial degradation, and survivin and caspase expressions were examined. In addition, global protein expression profiles were analyzed using proteomic analysis. Results demonstrated significant decrease in proliferation and increase in apoptosis in cells exposed to farnesol and C. albicansculture media. Several regenerative therapies have been used for maxillary sinus grafting. However, recent advances in modern bone tissue engineering techniques have been evaluated. The aim of this histologic report was to evaluate the bone obtained by a culture of autogenous osteoblasts seeded on polyglycolic-polylactid scaffolds in maxillary sinus augmentation. REFERENCES 1. Greenwood, Slack and Peutherer. Medical microbiology;15th edition. 2. Russel AD et al 1992. Principles and practice of disinfection, sterilisation and preservation. 2nd edi. Oxford: blackwill scientific. 3. Satish Gupta. Short textbook of Microbiology; 7th edition. 4. Ananthnarayan, Paniker. Textbook of th Microbiology; 6 edition. 5. Mackie, McCartney. Practical medical th microbiology; 14 edition. 6. Condayan C. Culture media. Nature Reviews Microbiology 2008, 6:646.

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