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Culture Media Microbiology Presentation.pptx

This document explains the various Culture Media

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CULTURE MEDIA
Culture Media  Liquid or solid substance - contains nutrients to support the growth, and survival of microorganisms
Constituents of Culture Media Water and Electrolytes Peptone : Mixture of partially digested proteins - obtained from various sources - heart muscle, casein or fibrin, or soya. Agar : Used for solidifying the culture media Solid Media : 1-2% Agar, Semi solid Media : 0.5% Meat extract Yeast extract Blood and serum : Important components of enriched media; provide extra nutrition to fastidious bacteria
Types of Culture Media Based on consistency, culture media are grouped into:  Liquid (or broth) media  Semisolid media  Solid media
Types of Culture Media Conventional culture media: Prepared from nutrients - aqueous extract of meat, peptone.  Simple/basal media  Enriched media  Enrichment broth  Selective media  Differential media  Transport media  Anaerobic media. Automated culture media: MaiMainly available for blood and sterile body fluid culture. Based on the growth detection, culture media are classified as
Simple/Basal Media  Contain minimum ingredients that support the growth of non-fastidious bacteria.  Peptone water: Contains peptone (1%) + NaCl (0.5%) + water  Nutrient broth: Peptone water + meat extract (1%).  Nutrient agar: Nutrient broth + 2% agar  Semisolid medium: Concentration of agar - reduced to 0.2–0.5 %.
Uses of Basal Media Testing the non-fastidiousness of bacteria. Serve as base for the preparation of many other media. Performing the biochemical tests, such as oxidase, catalase, etc. Study the colony morphology Pigment demonstration.
Enriched Media  To Basal Media Substances such as blood, serum or Egg are added  Used to grow bacteria which require extra nutrients  Eg : Blood Agar, Chocolate Agar Indole Biochemical test Blood Agar Chocolate Aagar
Enriched Media (Cont..) Blood agar Prepared by adding 5-10% of sheep blood to the molten nutrient agar at 45°C. Tests the hemolytic property of the bacteria, which may be either- i) partial or α (green) hemolysis and ii) complete or β hemolysis
Enriched Media (Cont..) Chocolate agar: Heated blood agar, prepared by adding 5 -10% of sheep blood to the molten nutrient agar at 70°C Supports certain highly fastidious bacteria - Haemophilus influenzae that does not grow on blood agar
Enrichment Broth Liquid media added with inhibitory agents which selectively allow certain organism to grow and inhibit others. Important for isolation of pathogens from clinical specimens which also contain normal flora (e.g. stool and sputum specimen). Examples : Tetrathionate broth—Used for Salmonella Typhi Selenite F broth—Used for Shigella
Selective Media Solid media containing inhibitory substances - inhibit the normal flora present in the specimen and allow the pathogens to grow Example : Lowenstein Jensen (LJ) medium : Mycobacterium tuberculosis DCA (Deoxycholate Citrate Agar) : Salmonella and Shigella from stool
Selective Media Lowenstein–Jensen medium Lowenstein–Jensen medium
Transport Media Used for the transport of the clinical specimens suspected to contain delicate organism or when delay is expected while transporting the specimens from the site of collection to the laboratory Bacteria do not multiply in the transport media - they only remain viable. Example Alkaline peptone water for Vibrio cholerae Amies medium, Stuart’s medium for Neisseria
Differential Media Differentiate between two groups of bacteria - by using an indicator Example MacConkey agar : Differentiates organisms into LF (pink colonies, e.g. Escherichia coli) and NLF or (colorless colonies, e.g. Shigella).
Anaerobic Culture Media The Anaerobic media contain reducing substances which take up oxygen and create lower redox potential - permit the growth of obligate anaerobes, such as Clostridium Robertson’s cooked meat (RCM) broth
Blood Culture Media Recovery of bacteria from blood is difficult - they are usually present in lesser quantity and many of the blood pathogens are fastidious. Therefore, enriched media - used for isolating microorganisms from blood. Available either as conventional or automated media
Conventional Blood Culture Media Two types: Monophasic medium: Contains brain–heart infusion (BHI) broth. Biphasic medium: Liquid phase containing BHI broth and a solid agar slope made up of BHI agar
Automated Blood Culture Techniques Continuous automated monitoring: Blood culture bottles - periodically monitored for the microbial growth - every 10 minutes by the instrument. BacT/ALERT 3D: When bacteria multiply - produce CO2 - increases the pH - changes the color of a blue-green sensor present at the bottom of the bottle to yellow
CULTURE METHODS
CULTURE METHODS Involve inoculating the specimen on to appropriate culture media, followed by incubating the culture plates in appropriate conditions. Bacteriological loop and straight wire B. Flaming the loop (red hot)
Biosafety Cabinet (BSC) It is an enclosed, ventilated laboratory work station, used to protect the laboratory personnel while working with potential infectious clinical specimens.
Streak Culture
Liquid Culture Used for culture of specimens - blood or body fluids - inoculated by directly adding the specimen in to the liquid medium or with the help of a syringe or pipette. Bacterial growth - detected by observing turbidity in the medium. Some aerobic bacteria form surface pellicles
Lawn or Carpet Culture Uniform lawn of bacterial growth is obtained by either swabbing or flooding with a bacterial broth onto the culture plate. Useful to carry out antimicrobial susceptibility testing (AST) by disk diffusion method.
Stroke Culture Carried out on agar slopes or slants by streaking the straight wire in a zigzag fashion. Used for biochemical test such as urease test.
Stab Culture Made by stabbing the semisolid agar butt by a straight wire. It is Used for motility testing using mannitol motility medium.
Incubatory Conditions Most pathogenic bacteria - aerobes or facultative anaerobes - grow best at 37°C. Inoculated culture plates - incubated at 37°C aerobically for overnight in an incubator. Incubator
Other Incubatory Conditions For capnophilic bacteria: Candle jar is used Inoculated media - placed inside a jar with a lighted candle - burning candle reduces oxygen to a point where the flame goes off. Provides an atmosphere of approximately 3–5% CO2 . Useful for capnophilic bacteria - Brucella, Streptococcus, pneumococcus and gonococcus
Anaerobic Culture Methods Obligate anaerobic bacteria - grow only in the absence of oxygen. Anaerobic culture methods includes: Evacuation and Replacement Absorption of Oxygen by Chemical Methods Anaerobic Glove box and Anaerobic Work Station Reducing Agents
Evacuation and Replacement Involves evacuation of the air from jar and replacement with inert gas like hydrogen followed by removal of the residual oxygen by use of a catalyst. It is Carried out either by: Manual method – using McIntosh and Filde’s anaerobic Automated system (Anoxomat)
Evacuation and Replacement (Cont..) Manual method- McIntosh and Filde’s anaerobic jar - most popular method for creating anaerobiosis in the past, now not in use.
Evacuation and Replacement (Cont..) Automated system (Anoxomat): Automatically evacuates air - replaces by hydrogen gas from a cylinder. Catalyst - sachet containing aluminum pellets coated with palladium
Absorption of Oxygen by Chemical Methods GasPak system Traces of oxygen - removed by same catalyst used for Anoxomat.
Reducing Agents Oxygen in culture media - reduced by various reducing agents - glucose, thioglycollate, cooked meat pieces, cysteine and ascorbic acid. Robertson cooked meat broth - most widely employed anaerobic culture medium - uses chopped meat particles (beef heart) as reducing agent
Colony Morphology After overnight incubation - culture media are removed from the incubator - examined under bright illumination. Hemolysis on Blood Agar Partial or α hemolysis Complete or β hemolysis No hemolysis (γ hemolysis, a misnomer)
CULTURE IDENTIFICATION Identification of bacteria from culture is made either by: Conventional biochemical tests or Automated identification systems.
Biochemical tests Examples catalase and oxidase tests For gram-negative bacilli - Common biochemical tests done routinely – ‘ICUT’ Indole test Citrate utilization test Urea hydrolysis test Triple sugar iron test (TSI).
Indole Citrate Urease Catalase Test TSI Test Oxidase test
Biochemical tests For gram-positive cocci: Coagulase test (for Staphylococcus aureus) CAMP (Christie-Atkins-Munch-Petersen) test - group B Streptococcus. Bile esculin hydrolysis test (for Enterococcus) Inulin fermentation (for pneumococcus) Bile solubility test (for pneumococcus)
Coagulase test CAMP test Bile Solubility Test BEA test
Automated Systems for Bacterial Identification MALDI–TOF VITEK 2 Phoenix
ANTIMICROBIAL SUSCEPTIBILITY TEST
Antimicrobial Susceptibility Test Bacteria exhibit great strain variations in susceptibility to antimicrobial agents - AST plays a vital role to guide the clinician for tailoring the empirical antibiotic therapy to pathogen-directed therapy. Performed only for pathogenic bacteria isolated from the specimen, and not for the commensal bacteria. Classified into phenotypic and genotypic methods.
Antimicrobial Susceptibility Test (Cont..) Phenotypic methods - Disk Diffusion Method- e.g. Kirby–Bauer’s disk diffusion (DD) test Dilution tests: Broth Dilution and agar dilution Method Epsilometer or E-test Automated AST-e.g. Vitek, Phoenix and Microscan systems Genotypic methods - PCR detecting drug resistant genes
Kirby–Bauer’s disk diffusion Method Antibiotic disks - impregnated on to a suitable medium lawn cultured with the test isolate. Mueller–Hinton agar (MHA) - standard medium used for AST Procedure Directly suspending the colony in the normal saline or Inoculating into suitable broth and incubating at 37°C for 2 hours
AST Procedure (Cont..) Lawn culture: Broth - inoculated on to the medium by spreading with sterile swabs Disks impregnation: Disks - placed atleast 24 mm (center to center) apart on the MHA plate. Maximum up to 6 disks can be applied on a 100 mm plate.
Interpretation Susceptibility to the drug - determined by the zone of inhibition of bacterial growth around the disk - measured by Vernier caliper.
Dilution Tests Antimicrobial agent - serially diluted - each dilution is tested with the test organism for ASTand the MIC is calculated. MIC (minimum inhibitory concentration) - lowest concentration of an antimicrobial agent that will inhibit the visible growth of microorganism after overnight incubation.
Dilution Tests (Cont..) Depending upon whether the dilutions of the antimicrobial agent are made in agar or broth, there are two types of dilution tests. Broth Dilution Method Agar Dilution Method
Broth Dilution Method Two types: Macro broth dilution (performed in tubes) Micro broth dilution (performed in microtiter plate).
Broth Dilution Method (Cont..) Serial dilutions of the antimicrobial agent in Mueller Hinton broth are taken in tubes - each tube is inoculated with a fixed amount of suspension of the test organism. MIC - determined by - lowest concentration of the drug at which there is no visible growth, i.e. broth appears clear
Broth Dilution Method (Cont..)
Broth Dilution Method (Cont..) Minimum bactericidal concentration (MBC) - obtained by subculturing from each tube (showing no growth) onto a nutrient agar plate without any antimicrobial agent. Tube containing the lowest concentration of the drug that fails to show growth, on subculture, is the MBC of the drug for that test strain.
Agar Dilution Method Serial dilutions of the drug - prepared in molten agar and poured into petri dishes. Test strain is spot inoculated. Advantage: Several strains can be tested at the same time by using the same plate Directly measures the MBC
Epsilometer or E-test Uses an absorbent strip containing predefined gradient (serial dilution) of antibiotic concentration immobilized along its length. Applied to a lawn inoculum of a bacterium. Antibiotic concentration at which the ellipse edge intersects the strip -MIC value.
Automated Antimicrobial Susceptibility Tests VITEK 2 identification and antimicrobial sensitivity system Phoenix System (Becton Dickinson) Micro Scan Walk Away system.
Molecular Methods (Detecting Drug-resistant Genes) PCR RT-PCR GeneXpert
SEROLOGY
SEROLOGY Detection of either antigen or antibody in the serum of the patient, by various immunological assays such as— Precipitation Agglutination ELISA Rapid test.
MOLECULAR METHODS for Lab Diagnosis PCR RT-PCR GeneXpert CB-NAAT
KEY POINTS
 Agar Used for preparation of Solid media-2%  Agar Used for preparation of semisolid media-0.5%  Blood Agar is prepared aby adding 5-10% of blood to Basal Media  β hemolysis is seen Observed in Blood Agar  Chocolate agar is prepared by adding blood to Molten nutrient Agar at 70°C  Enrichment Broth is Liquid media added with inhibitory agents which selectively allow certain organism to grow and inhibit others  Selective Media is a Solid Media added with inhibitory agents which selectively allow certain organism to grow and inhibit others
 Differential Media : Differentiate between two groups of bacteria - by using an indicator  Example for Differential Media : MacConkey agar  Example for Anaerobic Culture Media : Robertson’s cooked meat (RCM) broth  Biphasic medium [Bottle containing Brain Heart Infusion (BHI) broth & Agar slant ] is used for Blood culture  BacT/ALERT 3D is an Automated Blood culture System  Streak Culture is a common method used to isolate bacteria from agar medium  Lawn culture method used for Antimicrobial sensitivity testing
 Media used for Antimicrobial sensitivity testing is Mueller Hinton Agar  Most pathogenic bacteria - aerobes or facultative anaerobes - grow best at 37°C  Anaerobic culture media & methods are : McIntosh and Filde’s jar, Anoxomat method, Gas pack system method, RCM broth  Commonly used Anaerobic culture broth is RCM broth (Robertson cooked meat broth )  The methods used for Antimicrobial Suscpetibility testing are : Kirby bauer disk diffusion test, Broth dilution methos, Agar dilution method, Epilometer test (E-test )
What is the percentage of agar used for the preparation of solid media? 1) 0.1% 2) 30 % 3) 2 % 4) 50 %
What percentage of blood is typically added to basal media to prepare blood agar? 1) 0 % 2) 1 % 3) 5-10% 4) 30 %
How is chocolate agar prepared? 1) By adding chocolate chips to molten nutrient agar at 37°C 2) By adding blood to molten nutrient agar at 10°C 3) By adding blood to molten nutrient agar at 70°C 4) By adding cocoa powder to molten nutrient agar at 20°C
What is selective media? 1) Solid media with inhibitory agents 2) Liquid media with inhibitory agents 3) Solid media with growth-promoting agents 4) Liquid media with growth-promoting agents
Which of the following is an example of a commonly used differential media? 1.Nutrient agar 2.Blood agar 3.MacConkey agar 4.Chocolate agar
What is the purpose of using a biphasic medium? 1.To culture viruses 2.To culture fungi 3.To culture bacteria in anaerobic conditions 4.To culture bacteria for blood culture