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Laboratory Methods For Identification Of Bacteria. 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 infection After been grown on artificial nutrient media - PowerPoint PPT Presentation
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Laboratory Methods For Identification Of Bacteria
Laboratory MethodsFor Identification Of BacteriaBacteria are either identified inA 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 media
Bacteria are then identified byMicroscopic ExaminationExamination of fresh samples used for demonstration of bacterial motilityusing hanging drop methodMorphology and staining reactions of bacteria
The hanging Drop Method
Commonly used stains1- Simple stainse.g. methylene blue
2- Differential stainse.g. Gram stainPrimary stainMethyl violet (Crystal Violet)- Iodine mixtureDecolourizationAlcoholCounter stainDiluted carbol-fuchsin stain (Safranin)ResultsGram (+)PurpleGram (-)Red
Differencedue to structure of cell wall
Gram (+) Thick cell wallGram (-) Thin cell wall
A Gram stain of mixed Staphylococcus aureus
Grams StainDifferential Stain - divides bacteria into 2 groupsAcid FastNon Acid Fast
Used to identify organisms in the Genera Mycobacterium (high lipid and wax content in cell wall)
ZiehlNeelsen stainProcedureFix the smear of the specimen over the glass slideeither by heating or alcohol fixation
Pour carbol fuschin over smearheat gently until fumes appeardo not overheatallow it to stand for 5 minuteswash it off with water
Pour 20% sulphuric acid5%sulfuric acidis used for destainingMycobacterium lepraeinstead of the 20% used forMycobacterium tuberculosiswait for one minutekeep on repeating this step until the slide appears light pink in colorwash off with water
Pour methylene bluewait for two minutesagain wash with water
Allow it to air dryexamine under oil immersion lens
ResultAcid Fast organismRed as Mycobacterium tuberculosisNon Acid Fast organismBlue as Enterobacteriaceae family
Mycobacterium tuberculosis(stained red) in tissue (blue)
A. Non Acid-fast bacteria B. Acid-fast bacteriaSpecial stainsCapsule stain and Flagella stain
Encapsulated Bacillus sp. stained using Maneval's capsule staining method
Pseudomonas fluorescens cultured on nutrient agar, stained usingthe Presque Isle flagella stain
(II) Cultural CharactersBacteria need nutritive culture media to multiply in vitro
An 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 growth 2- Water 3- Various salts needed for bacterial growth
Defined media (also known as chemically defined media or synthetic media)Classification of MediaMedia can be classified into
1-Minimal media ( simple medium)It contains the basic nutritive requirementse.g. nutrient broths and agar media
2- Selective mediaSelective media are used for the growth of only selective microbes
It contains antibiotics, dye, or specific chemicalsinhibits the growth of most types of microbestimulate the isolation of one type
Mannitol salt agar (MSA)selective for Gram positive (+ve) bacteria
An MSA plate with Micrococcus sp. (1), Staphylococcus epidermis (2) and S. aureus colonies (3).Blood-free, charcoal-based selective medium agar (CSM)isolation of Campylobacter sp.
Blood-free, charcoal-based selective medium agar (CSM) for isolation of Campylobacter.LwensteinJensen mediumenriched selective media for T.B.
Lwenstein-Jensen medium used for growing M. tuberculosis in a McCartney bottle
Distinctive clusters of colorless Mycobacterium tuberculosisTCBS agar (Thiosulfate-citrate-bile salts-sucrose agar)selective for Vibrio cholerae due to alkaline pH
Yellow coloured (sucrose fermenting) colonies of Vibrio cholerae on TCBS agar.3-Differential mediaDifferential media or indicator mediadistinguish one microorganism type from another growing on the same media
Indicatorsneutral redphenol redeosin Ymethylene blue
Examples of differential media include
Eosin methylene blue (EMB)differential for lactose and sucrose fermentation
E. coli on EMB agarMacConkey (MCK)differential for lactose fermentation
A MacConkey agar plate with an active bacterial culture
4- Enriched media Enriched media contain the nutrients required to support the growth of a wide variety of organismsincluding some of the more fastidious ones
Blood agarIs an enriched medium in which nutritionally rich whole blood supplements the basic nutrientsIt contains 5-10% human or animal blood
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)Chocolate agar (heated blood agar)enriched with heat-treated blood (40-45C).
Comparison of two culture media types used to grow Neisseria gonorrhoeae bacteriaLofflers serum mediaHorse serum + glucose in a ratio 3:1It is used for cultivation of Corynebacterium diphtheriae
5- Transport mediaTransport medium is a simple organic mediummaintain the viability of all organisms in the specimenwithout altering their concentration
This type of medium mainly used for temporary storage of specimensbeing transported to the laboratory for cultivation
Examples of transport media includeThioglycollate broth for strict anaerobes
Thioglycollate broth medium is recommended to isolate strict anaerobes should an anaerobic infection be suspected
The colonial appearance on culture mediaShapeThe colonies may be small (pin-point) fimbriate, flat or convex
Colour
The colonies may be colorless or bacteria produce endopigments which give the colonies a characterestic colour
Staph. aureus produce golden yellow coloniesStaph. albus produce white endopigmentStaph. citreus produce a lemon yellow endopigment
The bacteria may produce exopigmentsPseudomonas aeruginosa produce a green exopigments in the surrounding mediaAntimicrobial ChemotherapyAnantibacterial agentis a compound or substance that kills or slows down the growth ofbacteria
Antibiotic(s) has come to include a broader range ofantimicrobialcompounds, includinganti-fungal and other compounds
It is produced by microbes and is harmful to other microbes, except viruses
These includebeta-lactam antibacterialpenicillin(produced by Penicillium notatum)cephalosporin
Compounds that are still isolated from living organismsAminoglycosides
Other chemotherapeutic agents produced by chemical synthesisSulfonamidesQuinolones
Classification of AntibioticsAccording to agent action
Antibacterial agents are divided into two broad groups based on their biological effect on microorganisms
bactericidalagents kill bacteriabacteriostatic agentsslow down or stall bacterial growth
Bactericidal antibiotics
Antibiotics that inhibit cell wall synthesisBeta-lactam antibioticspenicillin derivatives, andcephalosporins
Aminoglycosidic antibiotics are usually considered bactericidalalthough they may be bacteriostatic with some organisms
Bacteriostaticantibioticslimit the growth ofbacteriaby interfering withbacterialproteinproductionDNAreplicationOr other aspects of bacterial cellularmetabolism
This group includesTetracyclinesSulphonamidesTrimethoprimChloramphenicolMacrolides
Antibiotic sensitivity testAntibiotic sensitivityis a term used to describe the susceptibility ofbacteriatoantibiotics
Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infectionin vivo
Testing for antibiotic sensitivity is often done by theKirby-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 forMinimum Inhibitory Concentrationdetermination
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.
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.The Dilution MethodSerial dilutions of antibiotics are incorporated in agar containing or broth culture media
The lowest concentration of antibiotic that prevents visible growth after an 18-24 hours incubation period is known as minimal inhibitory concentration (MIC)
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 media
The lowest concentration of antibiotic that totally suppresses growth after overnight incubation is known as MBC
Minimum Inhibitory Concentration
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