CULTURE MEDIA AND METHODS

Dr.Shruthi.k.patil

REFERENCES
Laksman Samarnayak-Microbiology for dentistry .3 edition Mackie and mcCartney- Practical medical microbiology Godkar-Text book of medical laboratory technology.2 edition Bailey and Scotts-Diagnostic medical microbiology.9 edition Anathnarayan & Paniker-Text book of microbiology.7 edition Chakraborthy -Textbook of microbiology

INTRODUCTION
It is essential to grow the organisms from infected material to identify the cause of infection. Only after growing them in pure culture,it is usually possible to identify them. For studying their characteristics, it is necessary to culture them.

 Majority of the bacteria to be studied are pathogenic. Hence to obtain suitable growth of the bacteria, culture media should approximate to the composition and reaction of the tissues and body fluids in which the bacteria grow. No single medium can satisfy all requirements. A medium could perfect only for a limited range.

LABARATORY METHODS AND TECHNIQUES

Non cultural methods. Immunological methods. Cultural methods:Solid and liquid media used for bacterial and fungal growth. Cultured cells derived from animals and humans used for viral growth.

 Louis pasteur introduced the use of complex media. Chicken broth ,Urine and Meat broth were in use. All were liquid media. Growth could be obtained but growth characteristics and purity of the culture could not be made out. Robert koch introduced solid media. He used gelatin. Gelatin melts at 24 degrees and often gets liquefied by the growing organisms . Hence it was found unsatisfactory. On the suggestion of Frauhesse, he introduced agar .

DEFINITION
Culture medium:- A liquid or a gelatinous substance containing nutrients in which microorganisms or tissues are cultivated for scientific purposes.

INDICATIONS FOR BACTERIAL CULTURE

Isolation of the organisms in pure culture from clinical specimens and their final identification Antimicrobial drug sensitivity tests. Preparation of antigens to be employed in serodiagnosis of infective diseases. Preparation of vaccine.

Characteristics of an ideal culture medium

It must give a satisfactory growth from a small inoculum and ideally from a single cell It should give a rapid growth possible for all characteristics Easy to prepare Reasonably cheap Easily reproducible Make it possible for all characteristics.

BASIC REQUIREMENTS OF CULTURE MEDIA

Energy source Carbon source Nitrogen source Salts Satisfactory pH 7.2-7.6 Adequate oxidation and reduction potential Growth factors:- eg Tryptophan for s. typhi Glutathion for gonococci

PH
Media should be adjusted to the ph optimal to the growth of the organism. Pathogenic bacteria grow best around 7.3 i.e., alkaline Ph. streptococcus pneumonia -7.8 range 6.5-8.3 E.coli-6.5 range 4.4-7.8 yeast and fungi acid medium

Buffers
Important to keep the ph within the range. Most of the microorganisms produce acids and alkalis by their metabolic activities this must be prevented from altering the ph of the environment too radically. For eg: when bacteria grown on medium containing sugar produce acids, these acids accumulate in unbuffered medium organism would be killed by the low ph produced.

Oxidation -reduction potential

Redox conditions in a medium are very important in the growth of certain bacteria. Strict aerobes are able to grow only in the presence of dissolved oxygen while strict anaerobes require reducing conditions and hence the absence of dissolved oxygen.

Temperature
Incubation of culture at 37c is standard practice in the culture of pathogenic bacteria . Camphylobacter 43c Laptospires 30c Fungi 25-28c Some fungi 22c

Constituents of bacteriological media

water: low mineral content. Agar:variety of seaweeds In powder form Long chain polysaccharides, inorganic salts, protein like material, long chian fatty acids. melts at 95 c and solidifies at 42c. serum

 Growth enriching constituents e.g.:meat extract yeast extract malt extract Blood: defibrinated horse or sheep blood is used.

Collection and transport of specimen

Provision for clinical information : Appropriate tests for each specimen to be selected by microbiologists according to clinical information given in accompanying request form. Hence information such as age, main clinical condition, date of onset of illness, recent/current antibiotic therapy, antibiotic allergies and history of previous specimens are all important for rationalization of investigations and should be supplied with the specimen.

 Specimen should be as fresh as possible . e.g.- Viruses and anaerobes Staphylococci and coliforms Transport specimen should be in an appropriate medium. Specimen for bacteriological investigations should be forwarded as soon as possible to the laboratory in robust, leak proof sterile container.

TRANSPORT MEDIUM
When specimen like faeces, throat swab etc are to be sent to laboratary from distant places, the pathogenic bacteria may not survive time taken for transmit or may overgrown by non pathogenic bacteria for transporting specimens. Helps to maintain the viability of the organisms

Transport media
Bacteriological transport media: semi solid non nutrient agar such as the Stuart transport media is widely used for urethral discharge(gonococci). It maintains the viability of the Gonococci. Composition:-sodium thioglycollate And glycerophosphate, cacl2, agar, Methylene blue and distilled Water.

 Glycero saline transport medium for stool(typhoid bacilli):- prevents the intestinal organism over growing the typhoid bacilli.

Bile peptone transport medium for stool(cholera vibrios).

 Amies transport medium:Modification of Stuart's transport medium used to preserve the viability of anaerobes and other pathogens

 Pikes medium:- used to preserve Streptococcus pyogenes, Pneumococci and Haemophilus influenzae in nose and throat swabs.

Carry Blair transport medium:used to preserve salmonella, shigella and vibrio.

Sterilization of medium
Clean but unsterile glassware is used. Medium and container are together sterilized by heat. Liquid media distributed in test tubes and the test tubes should be half filled. Small screw capped bottle are almost completely filled. Large bottles should not be filled more than 75-80% .

Sterilization of prepared medium

Autoclaving is the best method of sterilization. Heat penetration and sterilization time, varies greatly with volume of medium and also with container. For test tube containing 10ml of medium, sterilization time -15 min at121c or 35 min at 115c. Molten agar require same sterilization time as liquid media, if agar is solid 5-10mins must be added for melting.

 Tubes and the bottles of the medium must not be held in air tight tins . Wire crates are suitable. Heat sensitive ingredients such as blood, serum, egg yolk can be obtained sterile from commercial sources other can be sterilized by bacterial filters.

DISTRIBUTION OF THE STERILISED MEDIA WITH STERILE PRECAUTIONS

LIQUID MEDIA: Ingredients stable to heat are prepared and sterilized, unstable ingredients added with sterile precautions and then medium is distributed with sterile precautions into sterile containers.

SOLID MEDIA :-

Tubing and bottling of solid media:Solid media may be distributed in tubes or bottles, this is done for storage. If it is used for direct culture in a test tube or bottle, shape in which the medium is allowed to solidify depends on the method of inoculation for which it is used.

Pouring plates: Solid media in petridishes are usually referred to as plates. Dish of 90mm diameter, 14ml of medium usually ample. Plates are poured with sterile precautions. Melted medium poured into the dishes are left undisturbed until the medium as set.

Surface of the medium should be dry.

Types of media
1. Solid media Liquid media and Semisolid media. 2. Aerobic media and Anaerobic media 3. Simple media Complex media and Special media

Enrichment media Enriched media Selective media Differential media Indicator media

Solid media

Simple solid media Nutrient agar

Complex solid media- Blood agar Chocolate agar Loefflers serum Dorsets egg

Mac konkey agar Lowenstein Jensen media

Objectives of solid media

Discrete colony forming units, single pure colonies can be picked from primary plate for subculture on secondary plate. Observation of colonial characteristics helpful in identification of bacteria. Quantification of organisms as colony forming units(CFU).

Liquid media
Simple liquid media Peptone water Nutrient broth Complex liquid media:Tetrathionate broth Selenite F broth Thiogylcolate broth Alkaline peptone water Robertsons cooked meat media

OBJECTIVES OF LIQUID MEDIA

Growth of small no. of bacteria present in specimen with antibiotics. Antibiotics diluted in fluid medium ,promots growth of organism. Promoting the growth of the specific bacterium while supressing the other bacteria. Test the biochemical activities of the bacteria for identification purposes.

DISADVANTAGES OF LIQUID MEDIA

Growth could not exhibit specifically characteristic appearance. Organisms cannot be separated.

SIMPLE MEDIA
MEDIUM NUTRIENT BROTH PEPTONE WATER COMPOSITION Peptone, meat extract, sodium chloride, water Peptone, sodium chloride, water Agar and nutrient broth USE Routine culture Routine culture, basal media for sugar fermentation test Routine laboratory diagnosis.

NUTRIENT AGAR

 In nutrient agar if conc. of agar reduced 0.2-0.5%, sloppy agar obtained which enables the motile organisms to spread.

In increased conc. of agar to 6% spreading of organisms.

COMPLE X MEDIA
A media other than simple media is complex media. All special media are complex media.

SPECIAL MEDIA
Enriched media: Substances such as blood, serum and egg are added to a basal media. These media are used to grow bacteria which are more exacting in their nutritional needs. Examples:Blood agar chocolate agar Dorsetts Egg

BLOOD AGAR: Also an indicator medium, supports the growth of the ordinary bacteria. To detect and differentiate the hemolytic bacteria such as streptococcus pyogens. composition -nutrient agar, 5-10% sheep blood . Use:-General culture and streptococcus. .

 Preparation:-Transfer sterile nutrient agar to 50c water bath add aseptically sterile defibrinated sheep or horse blood. Mix gently and dispense in petridishes, ph should be adjusted to 7.3. incubate the plates at carbon di oxide enriched atmosphere at 37c overnight.

BLOOD AGAR

HEMOLYTIC PATTERNS: Alpha hemolysis:-green color around the colony(streptococcus pneumonia) Beta hemolysis:- a clear zone around the colony (streptococcus pyogens) Gamma hemolysis :- no hemolysis(streptococcus fecalis)

BETA HEMOLYSIS

 CHOCOLATE BLOOD AGAR:-It is an heated blood agar causing the medium to turn a chocolate brown color. Heat ruptures the red blood cells and liberates the nutrients . Ex:-Neisseria gonorrhaeae and Haemophilus species

 Preparation:-Melt the desired amount of nutrient agar, cool it in a water bath at 75c add 10% sterile blood and allow the medium to remain at 75c mixing blood and agar time to time until blood becomes chocolate brown color, within 10minutes,then pour as a slopes or plates.

 The bacterium to be isolated are overgrown by the unwanted bacteria usually the non pathogenic or commensal bacteria tend to overgrow the pathogenic ones. Substances are incorporated in the medium which have a stimulatory effect on the bacteria to be grown or an inhibitory effect on those to be supressed are incorporated in the medium. If Such substances are added to liquid medium results in absolute increase in the no. of wanted bacterium relative to the other bacteria. Ex:-S.typhi being overgrown by the E.coli in cultures from faeces.

ENRICHMENT MEDIA

 Examples:-Tetrathionate broth: Tetrathionate inhibits coliforms while allowing typhoid and paratyphoid bacilli to grow freely. Composition:-Nutrient broth sodium thiosulphate calcium carbonate iodine solution

 Add the calcium carbonate to the broth and sterilize it by autoclaving for 20 min at 121c.when it cools add thiosulphate, iodine and phenol red solutions with sterile precautions. Distribute 10ml in test tubes ideally medium should be used within a few hours of adding the iodine solution to the other reagents.

 Selenite F broth: For the isolation of salmonella species from fecal specimen and salmonella typhi from urine and also shigella. Composition:- peptone water , sodium Selenite

Thioglycollate broth: Used for the culture of anaerobic bacteria. Composition:-Nutrient broth Sodium thioglycollate 0.5% Sodium sulphite 0.1% Medium contains Methylene blue which acts as an oxidation reduction potential indicator which shows the medium is anaerobic.

Alkaline peptone water: Contains peptone water(ph 9.0) Culture of vibrio cholerae and other vibrio species. Alkaline nature of the medium inhibits the growth of fecal commensals while favors the multiplication of vibrios.

SELECTIVE MEDIA
It contains additives that enhance the growth of the desired organism by inhibiting the other organisms. Examples:Tellurite blood agar:-Corynebacterium diphtheriae Bile salt agar:-cholera vibrios Tcbs agar: vibrio cholerae and vibrio species MacConkeys agar:-Enterobacteria Lowenstein Jensen :-M.Tuberculosis SS Agar:-salmonella and shigella species. Mannitol salt agar:-S. aureus

 Ex:-Mac konkeys agar:- used for the cultivation of Enterobacteria. It contains neutral red to distinguish lactose fermenting coliforms from lactose non fermenting salmonella and shigella groups. Lactose fermenters produce pink colonies

 MacConkey's agar is a selective, differential plating medium which contains: Bile salts to inhibit (but not completely block) the growth of non-enteric bacteria .
Lactose as the sole carbon source

Neutral red indicator dye

Organisms which are able to ferment lactose produce acid as a result. This turns the neutral red indicator in the agar around the colony dark pink, like this Organisms which are not able to ferment lactose do not produce acid. The neutral red indicator in the agar around the colony remains colourless producing pale colonies, like this:

Lowenstein Jensen medium:used to isolate the mycobacterium tuberculosis and other Mycobacteria. Malachite green present in the medium prevents the growth of other organisms.

Salmonella-Shigella agar: This medium inhibitory to gram positive organism since it contains bile salts and also to coliforms since it contains brilliant green. Lactose fermenting organisms exhibit red and nonlactose organisms produce yellowish or colorless colonies. Salmonella produce hydrogen sulphide, which is indicated by black centers on the medium, due to the presence of ferric citrate.

 Mannitol salt agar: selective and indicator medium contains phenol red as indicator of acid production s. aureus and other staphylococci that ferment mannitol form colonies that turn the indicator yellow.

INDICATOR MEDIA
This media contains an indicator which changes a color when bacteria grows in them. Potassium tellurite in mcleods medium gets reduced to metallic tellurium by diphtheria bacillus to produce black colonies.

Ex:-Wilson and Blair medium:Incorporation of sulphite , s.typhi reduces sulphite to sulphide in the presence of glucose and colonies of s.typhi have a black metallic sheen.

DIFFERENTIAL MEDIUM
When the culture medium containing certain substances helps to distinguish differing properties of different bacteria, it is called differential media. It is an indicator medium contains:peptone, agar, lactose, sodium taurocholate and neutral red.lactose fermentors forms pink colonies while non lactose fermentors produce colorless or pale colonies. Ex:-Enterobacteria.

SUGAR MEDIUM
The standard sugar medium used for biochemical tests contain 1% sugar concerned in peptone water along with an indicator. A small tube is kept inverted in a large tube containing sugar media with the production of acid by bacteria, colorless medium turns pink and gas production is indicated by accumulation of gas bubbles on the top of inverted tube . Glucose, sucrose, lactose and mannitol .

FUNGAL CULTURE MEDIA

SABOURAUD DEXTROSE AGAR:Used to culture Candida albicans and other fungi. Composition:-Mycological peptone, agar, distilled water, glucose. Preparation:- dissolve the contents in distilled water. Ph should be 5.4-5.8. Sterilize at 121c for 15minutes and allow to cool to 50-55c, mix well and dispense in 15-20ml amounts in sterile petridishes and 7-10ml amounts in sterile tubes.

Sabaurauds agar

CULTURE METHODS
INDICATIONS:Isolate the bacteria in pure culture To demonstrate their properties Obtain sufficient growth for preparation of antigens and for other tests Determine sensitivity to antibiotics Estimate viable counts Maintain stock cultures

INSTRUMENTS USED TO SEED CULTURE MEDIA

Instruments is chosen according to the nature of the medium and inoculum . Inoculating wires such as nichrome are widely used. Nichrome is oxidizing hence either stainless steel or platinum iridium is a better choice to work with anaerobes. Wire is sterilized by holding vertically in a Bunsen flame until it becomes red hot.

 Loop- This is a flat circular and comletely closed loop of 2-4mm internal diameter mounted on a handle. It takes up a considerable amount of solid culture or a large drop of liquid. Straight wire- used for stab cultures and also for picking off single colonies. Thick wire- L shaped wire as a loop, more rigid and useful for lifting thick viscid sputum and holding the growths of single colonies.

 Scalpel- Making inoculations with scrapings from tissues and ulcers. Sterile pipettes- Graduated 1ml or 10ml glass pipettes or disposable glass or plastic pipettes are used for liquid inocula between 0.1 and 10ml. Sterile capillary pipettes- Used for sterile transfer of liquid in volumes that do not need to be measured accurately.

METHODS OF INOCULATION
Solid media:-Solid media are employed for isolation of the organism. These are dispensed in petridishes or tubes. Streak Lawn or carpet Stroke Stab Pour plate Sweep plate method

 Streak culture(surface plating): Routinely employed for the isolation of bacteria in pure culture from clinical specimens. Platinum loop is charged with the specimen to be cultured and transferred on to the surface of a well dried plate, on which it is spread over a small area at the periphery. Inoculum is then distributed thinly over the plate by streaking it with a loop in a series of parallel lines, in different segments of the plate.

 The loop should be flamed and cooled between the different sets of streaks. On incubation, growth may be confluent at the site of original inoculation, but becomes progressively thinner, and well separated colonies are obtained over the final series of streaks.

Lawn or carpet culture: Provides uniform growth of the bacterium useful for bacteriophage typing Antibiotic sensitivity testing (disc method) When large amount of growth is required Preparation of bacterial antigens and vaccines.

Preparation : Prepared by flooding the surface of the pate with a liquid culture or suspension of the bacterium, pippeting off excess inoculum and incubating the plate. Surface of the plate may be inoculated by applying a swab soaked in the bacterial culture or suspension.

 Stroke culture:Is made in tubes containing agar slope(slant) and is employed for providing a pure growth of the bacterium for slide agglutination and other diagnosis tests.

 Stab cultures; These cultures are employed mainly for demonstration of gelatin liquefaction and oxygen requirement of bacteria. Maintenance of stock cultures. Prepared by puncturing a suitable medium such as nutrient gelatin or glucose with a long straight, charged wire. Medium is allowed to set, with a tube in upright position, providing a flat surface at the top of the medium.

 Pour plate culture: It gives an estimate of the viable bacterial count in suspension and is recommended method for quantitative urine cultures. Tubes containing 15ml agar medium are melted and left to cool in a water bath at 45-50c. Appropriate dilutions of the medium are added to into sterile dishes and allowed to set. After incubation, colonies will seen well distributed throughout the depth of the medium and can be enumerated using colony counters.

Sweep plate method:-

Edges of the petridishes, containing the culture medium are rubbed over the fabric, with the medium facing it. The dust particles stirred up from the cloth settle on the culture medium, and colonies develop on incubation. They can be counted and estimates made.

Liquid cultures:- These culture can be inoculated in tubes, bottles or flasks by touching with a charged loop or by adding the inocula with pipettes or syringes. Large inocula can be employed in liquid cultures and hence this method is adopted for blood culture and for sterility tests, where the conc. Of bacteria in the inocula is expected to be small. Liquid cultures are prepared for the inocula containing antibiotics, as these are rendered ineffective by dilution in the medium.

ANAEROBIC CULTURE METHODS

By displacement of oxygen Cultivation in vacuum dessicator has been tried but proved to be unsatisfactory. Displacement of oxygen by inert gas like hydrogen or nitrogen is done from a sealed jar loaded with inoculated bacteria.

Widely used method is candle. Lighted candle placed in a air tight container loaded with inoculated plates. Burning candle use all the available oxygen inside before it gets extinguished, but in practice some amount oxygen always left behind.

 ABSORPTION OXYGEN BY CHEMICALS(CHEMICAL OR BIOLOGICAL METHOD)

1. Pyrogallic acid:- Buchner first introduced alkaline pyrogallol for anaerobiosis which absorbs oxygen. Pyrogallic acid is added to large tube containing solution of sodium hydroxide the tube is placed inside an air tight jar loaded with inoculated plates and tubes. 2. Mixture of powdered chromium and sulphuric acid:Two chemicals reacts in presence of available oxygen and produce chromous sulphate.

3.Gas-pak:-Disposable packet alluminium foil containing pellets of sodium borohydride and cobalt chloride and of citric acid and sodium bicarbonate widely used for preparing anaerobic jar. chemicals generate hydrogen and carbon di oxide inside the jar when water is added. Hydrogen combines with oxygen in presence of a catalyst present in under surface of lid jar. After inoculated plates are placed inside a large air tight jar, it is incubated 37c.

Biological methods:-

Anaerobiosis as been attempted by incubating the aerobic organisms along with anaerobic bacteria but the method is slow and ineffective.

By incorporating reducing agents in the media Oxygen in the culture media can be reduced by 1% glucose, 0.1% thioglycollate, 0.05% cystein, 0.1% ascorbic acid and cooked meat pieces. Two most widely employed anaerobic liquid culture media are:1. Thioglycollate broth:- contains nutrient broth and 1% thioglycollate.

Tube 1: Obligate Anaerobe note the absence of growth in the top portion of the broth where oxygen is present. Tube 2: Obligate Aerobe note the growth is only in the top portion of the tube where oxygen is present. Tube 3: Aerotolerant note the uniform growth from top to bottom. Tube 4: Facultative note the uneven distribution of growth from top to bottom (more growth at the top). Tube 5: Obligate Aerobe -- note the growth is only in the top portion of the tube where oxygen is present.

2. Robertson cooked meat medium: Consists of nutrient broth and pieces of fat free minced cooked meat of ox heart. It permits the growth of strict anaerobes and preserves delicate organisms. Unsaturated fatty acids present in meat utilize oxygen for autooxidtion, the reaction being catalysed by haematin in the meat. With the growth of saccharolytic anaerobes color of meat pieces turns to red while it becomes black in case of proteolytic anaerobes. Before inoculation, media are boiled in water bath at 80 c for hour to drive out oxygen.

By displacement and combustion of oxygen: Anaerobiosis obtained by McIntosh and Flides anaerobic jar is the most dependable and widely used method. Principle- spongy palladium or platinum kept inside the jar acts as a catalysing agent which causes slow combination of hydrogen and oxygen to form water.