Culture media

Culture Media And Culture Techniques Dr. Prabesh K Choudhary Resident, Dept of Pathology NAMS, Bir Hopspital: 

Culture Media And Culture Techniques Dr. Prabesh K Choudhary Resident, Dept of Pathology NAMS, Bir Hopspital

Why the Culture media ?: 

Why the Culture media ? (Diagnosis of diseases) Isolation and identification Maintenance of pure culture Biochemical characteristics Antibiotic susceptibility testing Molecular biological studies Mass cultivation for the production of Antibiotics: Vaccines: Anti-sera: Toxins:

Microbial Growth Requirement: 

Microbial Growth Requirement Similar environment and nutritional condition that exists in the natural environment Water (Distilled) a carbon source -structural organic molecules; energy a nitrogen source a phosphate source (DNA, RNA, ATP, Membrane) Mineral e.g. Fe, Mg and others

The Common Nutrient Requirements: 

The Common Nutrient Requirements Macro-elements (macronutrients) - C, O, H, N, S, P, K, Ca, Mg, and Fe - Required in relatively large amounts Micronutrients (trace elements) - Mn, Zn, Co, Mo, Ni, and Cu - Required in trace amount - Often supplied in water or in media components Organic growth factors - Vitamins, amino acids, purine, pyrimidine etc.

Culture media: Classification: 

Culture media: Classification Culture media are classified on the basis of their : - Physical state - Chemical composition - Function

Classification based on physical state:: 

Classification based on physical state: Broth/liquid medium Without a solidifying matrix/agent Growth determined by turbidity Eg. Nutrient Broth, MHB, TSB etc. Semisolid medium: Semisolid in state Low agar percentage (0.4-0.5 %) Eg: SIM, MIU Used as transport media and for bacterial motility and biochemical tests Biphasic medium: Eg. Castaneda medium

PowerPoint Presentation: 

Contd….. Solid medium [Agar] Polysaccaride extract from seaweed (solid at 1.5% w/v strength) High gelling action, setting temperature 32-39c, & melting temperature 90-95c Isolation of discrete colonies, identification informations, pure cultures Solid medium is generally made on : Slants Stabs Petri dishes

Blood culture bottles: 

Blood culture bottles

Basal Medium: 

Basal Medium Simple medium General purpose medium Commonly used for non-fastidious MOs, preparation of enriched media, stock culture, subculture before performing biochemical tests Eg : Nutrient agar, Nutrient broth etc.

Enriched Medium : 

Enriched Medium Basic media with addition of GF e.g. whole or lyzed blood, serum, peptones, yeast extract, vitamins, albumin etc. These may be added individually or in complex mixtures. Example of enriched medium: Blood agar , Chocolate agar . Required for H. Influenzae, N. Gonorrhoea, & some streptococci. *ENRICHMENT MEDIA: Fluid selective media e.g. Rappaport-Vassiliadis broth for Salmonella; used to increase the relative concentration of certain microorganisms in the culture prior to plating on solid selective medium

Selective Medium : 

Selective Medium Differential growth suppression: Are designed to suppress the growth of some microorganisms while allowing the growth of others (i.e. they select certain microbes). Supressed by: dyes, bile salts, antibiotics etc. Are solid so that individual colonies may be isolated. Examples : Mannitol salt agar (selects non-skin flora) S-S agar (selective for Salmonella and Shigella ) TMM (selective + enriched for Neisseria ) TCBS (selective for Vibrio cholerae ) and others

Selective Medium, Contd;: 

Selective Medium , Contd; Other ways of selection: - Anaerobic condition inhibits P. aeruginosa - Alkaline media for Vibrio cholera (TCBS) - Temperature: Listeria monocytogens(4c)

Differential medium: 

Differential medium Differential appearance: Allow growth of more than one microorganisms of interest but with morphologically distinguishable colonies. Any medium with a specific substrate and a good indicator can be used as a differential medium. Examples : Blood agar (various kinds of hemolysis) Mac Conkey agar (lactose fermentation = pink)

Transport Medium : 

Transport Medium Used for transport of specimens maintaining the viability of the organisms Prevents growth of commensals Protects the organisms from: Drying Change in pH Nutrition supply Examples: Stuart’s transport media Amies Transport media Cary-Blair transport media Anaerobic transport media Glycerol saline media

Common Transport Media and their Uses: 

Common Transport Media and their Uses Media: Uses: Amies transport media Pus/swabs Stuart’s TM STI pathogens Cary-Blair TM Enteric pathogens Bordetella TM B. pertusis Anaerobic TM Anaerobic bacteria Viral TM Viruses

Sterilization of culture media: 

Sterilization of culture media 1. Autoclaving: - Under-autoclaving: unsterile - Overautoclaving: precipitation, pH & destruction of essential components 2. Steaming at 100C: - Media containing ingredients inactivated or broken down over 100C e.g. Carry- Blair transport media (15mins) - Arnold & Koch steamer; Autoclave 3. Filtration: to sterilize additives that are heat sensitive e.g. serum & Sol containing urea Cellulose/membrane filter preferred

Sterility testing: 

Sterility testing Usually done for media to which blood or other substances are added after autoclaving Tubes & bottles: 5% of the batch at 35-37C overnight: turbidity/growth Petri dishes: best examined for contamination immediately before use

Pure Culture Technique: 

Pure Culture Technique Pure culture: It is a method of culturing microorganisms in which all of the individuals in a culture have originated from a single. Koch was the first to experimentally implemented the concept of pure culture This is carried out so as to: allow the characterization of types of microorganisms without the confounding presence of other

Inoculation of Culture Media: 

Inoculation of Culture Media Media should be checked for visual contamination or any change in appearance e.g. color Aseptic techniques must be used to prevent contamination

Aseptic Technique: 

Aseptic Technique Sterilize wire loops, straight wire & metal forceps before and after use Flame the necks of specimen bottles, culture bottles and tubes after removing and before replacing caps While inoculating, the caps should be hold in hands Always use racks to hold tubes and bottles Decontaminate the work bench before starting the day’s work and after finishing Protective clothings

Making a wire Loop: 

Making a wire Loop Must be made correctly to ensure inocula are well spread and to prevent the release of aerosols from long and springy loops Length of wire loop: 6cm Loop should be small (2mm) & fully closed Thickness of the wire: 26 or 27G Nichrome wire: reusable, cools quickly, not too rigid, & less expensive

Inoculation on solid Media; plate Culture methods: 

Inoculation on solid Media; plate Culture methods Petri plate is universally accepted container for solid culture medium. It is used for the study of colony characteristics of an organism . 30-40 mins incubation at 35-37C to dry the agar surface before use made up of plastic or heat resistant glass. - flat bottomed, shallow and 90 mm Plastic petri plates - disposable and cheaper . Glass petri plates - reusable but expensive.

Streaking: 

Streaking Streaking is a method of applying cultures to solid medium : a sterile loop is cooled and brought into contact with a culture the loop is then brought into contact with the surface of solid medium whereupon it is streaked (i.e., dragged) along the surface of the solid medium colonies grow along the points of the streak.

Illustration: 

Illustration

Inoculation of slopes: 

Inoculation of slopes Small amount of media is required e.g. Dorset egg medium, Loeffler serum It is used for maintenance of isolated bacteria or for performing biochemical tests. Use a sterile straight wire to streak the inoculum down the centre of the slope Then streak the inoculum in a zig-zag manner.

Inoculation of Stab Culture: 

Inoculation of Stab Culture To stab the medium, inoculate through the centre of the medium with a straight wire . Then withdraw the wire along the line of inoculum.

Inoculation of Liquid media: 

Inoculation of Liquid media Inoculation of nutrient broth and other liquid culture media are performed with the aid of wire loop or Pasteur pipette from colonial growth, liquid cultures or specimens. To subculture colonies into a liquid medium, pick the colonies with a sterile wire loop Rub the loop against the inner side of the tube below the level of the liquid medium. *All the inoculated media must be labeled with the identification no. of specimen and date of inoculation.*

Incubation of inoculated media: 

Incubation of inoculated media Delay in incubation affect the viability of pathogens Various factors play role: - Temperature - Humidity - Gasseous atmosphere: aerobic, anerobic, CO2 etc.

Temperature: 

Temperature Optimum, Minimum & Maximum temperature Most bacteria: 35-37C Yersinia enterocolitica: 20-28C Mycobacterium species: No growth of M. tuberculosis & M. ulcerans at 25C; oppurtunistic & saprophytic mycobacteria grow at this temperature

Humitidy: 

Humitidy Too dry atmosphere affect the viability of many pathogens e.g. gonnococci Inclusion of damp blotting paper in the bottom of the candle jar recommended for gonnococci

Gasseous atmosphere: 

Gasseous atmosphere Obligate aerobes: Pseudomonas aeruginosa Microaerophilic: Campylobacter jejunii Obligate anaerobe: Clostridia, Bacteriodes, anaerobic streptococci Facultative anaerobe: Staphylococcus aureus, Streptococcus pyogenes, E. coli Carboxuphilic: N. meningitidis

Anaerobic incubation: 

Anaerobic incubation Commercially produced O2 removing system: Sachets containing O2 consuming substrate in anaerobic jar or plastic bag Water is required for activation of sachet Merk Anaerocult anaerobic system: Iron powder, Citric acid, Sod. Carbonate & Kieselguhr Oxoid AneroGen system: Ascorbic acid

Anaerobic incubation; Contd.: 

Anaerobic incubation; Contd. Copper coated steel wool: Steel wool dipped into acidified CuSo4 sol into an air tight bag; Cu absorbs O2 Use of reducing agents: -Thioglycollate: Anaerobes in blood -Cooked meat medium: Clostridia & Bacteriodes; growth occur at bottom among meat particles (Reducing substances) Anaerobic container: Various types

Culturing in carbon dioxide: 

Culturing in carbon dioxide Reqired for N. gonorrhoea, N. meningitidis, Brucella & streptococcus pneumoniae Commercially available CO2 generating systems Use of white wax smokeless candle: 3-5% CO2 when the candle extinguish Chemical: reaction of NaHCO3 with tartaric acid or citric acid; Alka-Selter tablet Chemical methods not used for campylobacters

Examination of Culture: 

Examination of Culture Routinely used culture media MA ( McConkey Agar) BA ( Blood Agar) CA ( Chocolate Agar) NA ( Nutrient Agar) SS Agar ( Salmonella/Shigella Agar) After an overnight incubation (16-20hrs), the cultures are examined for visible bacterial growth.

Gross colony characters: 

Gross colony characters Types of colonies- round, irregular Size – mm Colour- White ,yellow, black Edge- distinct, curled Elevation-flat, raised, convex Surface- glistening, dull Density- Opaque, transparent Consistency- Butyrous, brittle, sticky Odor

Effect of the growth on the medium: 

Effect of the growth on the medium a) Hemolysis on BA - α, β & γ Hemolysis b) Pigment production - Yellow-green P. aeruginosa c) Changes in differential medium - Lactose fermentor / Non lactose fermentor