Presentation on theme: "Laboratory Methods For Identification Of Bacteria"— Presentation transcript:
1 Laboratory Methods For Identification Of Bacteria
2 Bacteria are either identified in A pathological specimen obtained from the patient(e.g. pus, sputum, urine, blood, stools, etc.)depending on the site of infectionAfter been grown on artificial nutrient mediaBacteria are then identified byMicroscopic ExaminationExamination of fresh samples used for demonstration of bacterial motilityusing hanging drop methodMorphology and staining reactions of bacteria
8 Ziehl–Neelsen stainDifferential Stain - divides bacteria into 2 groupsAcid FastNon Acid FastUsed to identify organisms in the Genera Mycobacterium (high lipid and wax content in cell wall)
9 Procedure Fix the smear of the specimen over the glass slide either by heating or alcohol fixationPour carbol fuschin over smearheat gently until fumes appeardo not overheatallow it to stand for 5 minuteswash it off with water
10 examine under oil immersion lens Pour 20% sulphuric acid5% sulfuric acid is used for destaining Mycobacterium leprae instead of the 20% used for Mycobacterium tuberculosiswait for one minutekeep on repeating this step until the slide appears light pink in colorwash off with waterPour methylene bluewait for two minutesagain wash with waterAllow it to air dryexamine under oil immersion lens
11 Result Acid Fast organism Non Acid Fast organism Red as Mycobacterium tuberculosisNon Acid Fast organismBlue as Enterobacteriaceae familyA. Non Acid-fast bacteriaB. Acid-fast bacteriaMycobacterium tuberculosis (stained red) in tissue (blue)
12 Special stains Capsule stain and Flagella stain Pseudomonas fluorescens cultured on nutrient agar, stained using the Presque Isle flagella stainEncapsulated Bacillus sp. stained using Maneval's capsule staining method
14 (II) Cultural Characters Bacteria need nutritive culture media to multiply in vitroAn undefined medium (also known as a basal or complex medium). It is a medium that contains:1- A carbon source such as glucose for bacterial growth2- Water3- Various salts needed for bacterial growthDefined media (also known as chemically defined media or synthetic media)
15 Classification of Media Media can be classified into1-Minimal media ( simple medium)It contains the basic nutritive requirementse.g. nutrient broths and agar media
16 2- Selective mediaSelective media are used for the growth of only selective microbesIt contains antibiotics, dye, or specific chemicalsinhibits the growth of most types of microbestimulate the isolation of one type
17 Mannitol salt agar (MSA) selective for Gram positive (+ve) bacteriaAn MSA plate with Micrococcus sp. (1), Staphylococcus epidermis (2) and S. aureus colonies (3).
18 Blood-free, charcoal-based selective medium agar (CSM) isolation of Campylobacter sp.Blood-free, charcoal-based selective medium agar (CSM) for isolation of Campylobacter.
19 Löwenstein–Jensen medium enriched selective media for T.B.Distinctive clusters of colorless Mycobacterium tuberculosisLöwenstein-Jensen medium used for growing M. tuberculosis in a McCartney bottle
20 TCBS agar (Thiosulfate-citrate-bile salts-sucrose agar) selective for Vibrio cholerae due to alkaline pHYellow coloured (sucrose fermenting) colonies of Vibrio cholerae on TCBS agar.
21 3-Differential media Differential media or indicator media Indicators distinguish one microorganism type from another growing on the same mediaIndicatorsneutral redphenol redeosin Ymethylene blue
22 Examples of differential media include Eosin methylene blue (EMB) differential for lactose and sucrose fermentationE. coli on EMB agar
23 MacConkey (MCK) differential for lactose fermentation A MacConkey agar plate with an active bacterial culture
24 4- Enriched mediaEnriched media contain the nutrients required to support the growth of a wide variety of organismsincluding some of the more fastidious onesBlood agarIs an enriched medium in which nutritionally rich whole blood supplements the basic nutrientsIt contains 5-10% human or animal blood
25 It shows the type of haemolytic activity of bacteria (complete, partial or non-haemolytic) Complete Haemolysis of RBCs (Beta Haemolytic Streptococci)Partial Haemolysis of RBCs (Alpha Haemolytic Streptococci)
26 Chocolate agar (heated blood agar) enriched with heat-treated blood (40-45°C).Comparison of two culture media types used to grow Neisseria gonorrhoeae bacteria
27 Lofflers serum media Horse serum + glucose in a ratio 3:1 It is used for cultivation of Corynebacterium diphtheriae
28 5- Transport media Transport medium is a simple organic medium maintain the viability of all organisms in the specimenwithout altering their concentrationThis type of medium mainly used for temporary storage of specimensbeing transported to the laboratory for cultivation
29 Examples of transport media include Thioglycollate broth for strict anaerobesThioglycollate broth medium is recommended to isolate strict anaerobes should an anaerobic infection be suspected
31 The colonial appearance on culture media ShapeThe colonies may be small (pin-point) fimbriate, flat or convexColourThe colonies may be colorless or bacteria produce endopigments which give the colonies a characterestic colourStaph. aureus produce golden yellow coloniesStaph. albus produce white endopigmentStaph. citreus produce a lemon yellow endopigmentThe bacteria may produce exopigmentsPseudomonas aeruginosa produce a green exopigments in the surrounding media
32 Antimicrobial Chemotherapy An antibacterial agent is a compound or substance that kills or slows down the growth of bacteriaAntibiotic(s) has come to include a broader range of antimicrobial compounds, including anti-fungal and other compoundsIt is produced by microbes and is harmful to other microbes, except viruses
33 Compounds that are still isolated from living organisms These includebeta-lactam antibacterialpenicillin (produced by Penicillium notatum)cephalosporinCompounds that are still isolated from living organismsAminoglycosidesOther chemotherapeutic agents produced by chemical synthesisSulfonamidesQuinolones
34 Classification of Antibiotics According to agent actionAntibacterial agents are divided into two broad groups based on their biological effect on microorganismsbactericidal agents kill bacteriabacteriostatic agents slow down or stall bacterial growth
35 Bactericidal antibiotics Antibiotics that inhibit cell wall synthesisBeta-lactam antibioticspenicillin derivatives, and cephalosporinsAminoglycosidic antibiotics are usually considered bactericidalalthough they may be bacteriostatic with some organisms
36 Bacteriostatic antibiotics limit the growth of bacteria by interfering with bacterial protein productionDNA replicationOr other aspects of bacterial cellular metabolismThis group includesTetracyclinesSulphonamidesTrimethoprimChloramphenicolMacrolides
37 Antibiotic sensitivity test Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibioticsAntibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo
38 Testing for antibiotic sensitivity is often done by the Kirby-Bauer method ( Disc-diffusion method) Other methods to test antimicrobial susceptibility include the E-test (also based on antibiotic diffusion)Agar and Broth dilution methods for Minimum Inhibitory Concentration determination
39 In Kirby-Bauer testing, white wafers containing antibiotics are placed on a plate of bacteria. Circles of poor bacterial growth surround some wafers indicating susceptibility to the antibiotic.
40 This is most commonly used in the setting of medicine, where a particular organism has been found to infect a patient, and the doctor treating the patient is seeking guidance on what concentration of antibiotic is suitable.
41 The Dilution MethodSerial dilutions of antibiotics are incorporated in agar containing or broth culture mediaThe lowest concentration of antibiotic that prevents visible growth after an hours incubation period is known as minimal inhibitory concentration (MIC)
42 The minimal bactericidal concentration (MBC) may be determined in broth dilution tests by subculturing the containers that show no growth on to antibiotic-free agar containing mediaThe lowest concentration of antibiotic that totally suppresses growth after overnight incubation is known as MBC