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