IDENTIFYING CLINICALLY RELEVANT MICROBES

Use Of Microscope:
.wet mount ,heat-fixed ,chemically fixed

WET MOUNT

Concentrated wet mounts of blood, urine or stool can be used to ascertain presence of eggs, cysts, lar vae, or even vegetative cells of parasites.

Detection of spirochaetes e.g. for Treponema pallidum (Syphilis), Borellia Burgdoferi (Lyme disease) is easier in phase-constrast and Dark field microscopy.

Acid-fast organisms like Mycobacterium tuborculosis are identified by flouroscence microscope. The flourochromes used are rhodamine and auramine.

Identification of fungi through hyphal structures. Fungal identification through morphology of reproductive structures like spores.

Gram staining and acid-fast staining fail to identify bacteria without cell-wall like Mycoplasmas.

IDENTIFICATION OF MOLDS

Lactophenol cotton blue(aniline) staining

Molds can be directly identified using fluoroscence.

10%Calcofluor White addition enhances identification

IMMUNOFLOUROSCENCE

.Direct .Indirect

Immunofluorescence is the technique in which flurochromes or fluroscent dyes are exposed to UV, violet or blue light to make them flouresce or emit visible light.

Dyes such as rhodamine B or FITC (Fluroscein iso thio cyanate) can be labelled to antibody molecule as well as antigen molecule very easily.

The pattern of fluroscence reveals antigens location.

Direct immunoflouroscence

Used for Group A Streptococci, enteropathogenic E.Coli, Neisseria meningititis, Salmonella typhimurium, Shigella sonnei, Listeria monocytogenes, Haemophilus influenza

Indirect Immunofluroscence is used for organisms like Treponema pallidum.

VIRAL IDENTIFICATION

.isolation in cell-culture .immunodiagnosis(ELISA) .radio immunoassay .latex agglutination .immuno-peroxidase test .molecular detection methods like nucleic acid probes and PCR amplification

DIFFERENT TYPES OF CELL CULTURES Primarily culture- the cells are derived directly from tissues Secondar y culture-sub-culture of primar y culture, finite life span

Continuous or immortal cell lines- e.g. HEp2 ; transformed cells generally epithelial cells; heteroploid

2-types of cell-culture detections for viruses1)Cytopathic effects obser vable morphological change that occurs in cells due to viral replication e.g. balooning, binding together, clustering and cell-death

2)Hemadsorption As virus causes changes in the plasma-membrane of infected cells; they can readily adsorb red blood cells

VIRAL REPLICATION IN EMBRYONATED EGG CELLSDifferent viruses grow in different cell-types of egg a)Allantoic cavity b)Amniotic cavity c)chorio-allantoic membrane

DETECTIONa)Pocks on chorio-allantoic membrane b)Hemagglutinins in the allantoic and amniotic fluids

DETECTION IN INOCULATED ANIMALS: .Signs of disease and death .Serological tests using mAB based immunoflouroscence viruses like cytomegalovirus, herpes simplex virus

DETECTION OF FUNGI

Slow culture; culture discarded after 30 days if no fungal colony appears. Culture conditions require at least one selective and one non-selective media. The colony characteristics are noted-morphology, color, dimorphism Serological examination suited for few fungi. Serological tests include complement fixation and immuno-diffusion FUNGI IDENTIFIABLE BY SEROLOGICAL TESTSBlastomyces dermatitidis Coccidioides imitis Histoplasma capsulatum Cr yptococcus neoformans (Cr yptococcal latex antigen test)

PARASITIC IDENTIFICATION Culture of parasites not viable. Identification by direct microscopic methods. Typical histological staining of blood; negative staining of other body-fluids and immunoflouroscence staining. Few serological tests are also available.

BACTERIAL IDENTIFICATION Isolation and growth/culture of microbe The time for visible growth to occur is an important variable in the clinical laboratory. Identification steps1)The source of specimen 2)Microscopic observation and gram reaction 3)The pattern of growth on selective, differential or metabolism determining media. 4) Special properties like hemolytic, metabolic and fermentative properties

IDENTIFICATION OF RICKETTSIAS AND CHLAMYDIAE Rickettsias can be diagnosed by immuno-assays or by isolation of the micro-organisms. Chlamydiae can be diagnosed by Giemsa staining, which detectsthe characteristic intracellular inclusion body. Immunofluroscence staining of tissues and cells with mABs is more sensitive. Nucleic acid hybridization and PCR based methods are still more sensitive.

IDENTIFICATION OF MYCOPLASMAS

Immunological (hemagglutinin) , complement-fixation, antigenantibody reactions using patients sera and PCR methods.

USE OF SELECTIVE MEDIA TO ISOLATE PURE BACTERIAL CULTURES

1)Salmonella-Shigella Agar Salmonella and Shigella which are lactose non-fermenters produce colorless colonies; bile salt inhibits many coliform growths; lactose fermenters produce pink colonies 2) Mannitol Salt (MS Agar) used for the identification of Staphylococci. The selectivity is obtained by the high salt conc.(7.5%) that inhibits growth of many bacteria. This media is also differential; pathogenic Staphylococci ferment mannitol to produce acid while non-pathogenic strains do not.

3)Bismuth sulfite (BS) agar Isolation of Salmonella enterica serovar Typhi, from stool, food specimens. This bacteria reduces sulfite to sulfide, resulting in black colonies with a metallic sheen. 4) The addition of blood, sera, or extracts to tryptic soy agar or broth will support growth of many fastidious bacteria. These media are used primarily to isolate bacteria from cerebrospinal fluid, pleural fluid, sputum and wound.

USE OF DIFFERENTIAL MEDIA-

EMB AGAR-Agar has lactose.Eosin methylene blue agar differentiates between lactose fermenters and lactose non-fermenters. E.Coli is a lactose fermenter, produces a dark colon or one that has a metallic sheen. lactose non-fermenters like salmonella produce colorless colonies. Mc Conkey Agar- The bile salts and cr ystal violet in this agar inhibits the growth of gram positive bacteria and fastidious gram-negative bacteria. Different lactose fermenting bacteria produce, different shades of red while lactose non-fermenters produce colorless colonies.

HEKTOEN ENTERIC AGAR- used to isolate shigella and salmonella; high bile salt concenothtrations

BLOOD AGAR- citrated blood added to tryptic soy broth Differentiation on the basis of hemolysis1)alpha-hemolysis greenish to brownish halo around colony- Streptococcus sps.( pneumoniae) 2)Beta- hemolysis clear zone around colony Staph.aureus and Streptococcus pyogenes 3)non-hemolytic- no change in the medium (staph. Strains)

SOME MEDIA FOR BIOCHEMICAL TESTS1) Urease broth-detect enzyme urease; some enteric bacteria can break down urea into ammonia and carbon-dioxide 2)TSI AGAR- contain lactose, sucrose and glucose; ferrous ammonium sulfate and sodium thio-sulfate. This agar identifies enteric organisms based on their ability to metabolize sugar and release sulfides from sulfate. 3)Citrate Agar- sodium citrate as carbon source and ammonium phosphate as nitrogen source; differentiates enteric bacteria on the basis of citrate utilization. 4)LIA AGAR- (lysine iron agar)-bacteria that can either deaminate or decarboxylate the amino-acid lysine. The media contains ferric ammonium citrate for detection of hydrogen sulfide release.

5)SIM AGAR- (Sulfide, indole, motility) medium- 3 different tests for enteric bacteria.

Recent microbial technologies include biosensors based on 1)micro-fluidic antigen sensors 2)Real time (20 mins) PCR 3)Spectroscopy 4)Liquid crystal amplification of microbial immune complexes API 20E system

BIOSENSORSIf the biosensor is integrated into computer microchip for information management has it is then called BIOCHIP. Biosensors that detect specific DNA sequences, expressed Proteins and metabolic products, have been developed- that use Optical (mostly fluoroscence), electrochemical or even mass Displacement, to report detection. The high degree of recognition required to reduce false positive Results is achieved by specific receptor like capture involved in Nucleic acid hybridization and antibody binding. Several microbial biosensors employ single-stranded DNA or RNA Sequences or antibody, for detection component.

Transducers used in biosensors1)Micro-cantilever systems detect the increased mass of the Receptor-bound ligand 2) surface acoustic wave device, detects change in specific Gravity 3)Bulk quartz resonator monitors fluid density and viscousity 4)Quartz crystal microbalance measures frequency change in Proportion to the mass of material deposited on the crystal. 5)micro-mirror sensor uses an optical fibre waveguide that Measures changes in reflectivity 6)Liquid crystal based system reports the reorientation of Polarized light.

The specific capture of a ligand and is reflected in the net change Measured by each system and results in a signal that announces the initial capture event. Microchip control of the primary and secondary signals has resulted in automation of the detection process.

BIOCHEMICAL TESTS1) CARBOHYDRATE FERMENTATION Acid or gas produced during fermentative growth -differentiates enteric bacteria as well as other genera or species 2) CASEIN HYDROLYSIS presence of caseinase that degrades milk protein; clear zone around the colonies Used to cultivate and differentiate aerobic actinomycetes based on casein utilization. E.g. Streptomyces Nocardia does not utilize casein

3)CATALASE TEST Detects the presence of catalase; converts hydrogen peroxide to water and oxygen. Differentiates- Staph and strepto-enti Bacillus from clostridium 4)CITRATE UTILIZATION citrate used as sole carbon source; alkalization of the medium Klebsiella, Enterobacter, Salmonella (+) Escherichia, Edwardsiella (-) 5)COAGULASE causes plasma to clot; Staph. Aureus (+) Staph. Epidermis (-) 6)DECARBOXYLASES- act on amino-acids- R, K and ornithine release of carbon-dioxide identification of enteric bacteria

7)ESCULIN HYDROLYSIS Tests for the cleavage of a glycoside Staph. Aureus (-) Enterococci and S.mutans (+) 8)Beta- galactosidase test (ONPG TEST) breakdown of lactose to glucose and galactose Salmonella (-), citrobacter (+) and identification of pseudomonads. 9)GELATIN LIQUEFACTION presence of proteases that hydrolyze and liquify gelatin identification of Clostridium, Serratia, Pseudomonas and Flavobacterium 10)HYDROGEN SULFIDE Detects formation of hydrogen sulfide from amino-acid cysteine; action of cysteine desulfurase Identification of Edwardsiella, Proteus and Salmonella

11)IMViC The indole test detects the production of indole from the amino-acid tryptophan. Methyl red is a pH indicator to determine Whether the bacterium carries out mixed fermentation. VP(Vogues Proskauer) test detects the production of acetoin The citrate test determines production of acetoin. The citrate Test determines, whether or not the bacterium utilize sodium Citrate as a sole source of carbon. Escherichia MR (+), VP (-), Indole (+) Enterobacter- MR (-). VP(+), Indole(-) Klebsiella pneumonia-MR (-). VP(+), Indole(-) Also used to characterize members of the genus Bacillus 12) LIPID HYDROLYSIS Detects the presence of lipase; which breaks down lipids into Simple fatty-acids and glycerol. Used in the separation of clostridia.

13)NITRATE REDUCTION Detects whether a bacterium can use nitrate as an electron acceptor Used in the identification of enteric bacteria which are usually (+) 14) OXIDASE Detects the presence of cytochrome c oxidase that is able to reduce oxygen and artificial electron acceptors Neisseria, Moraxella (+) Acinetobacter (-) and enterics (-), Pseudomonads (+) 15) PHENYL-ALANINE DEAMINASE Deamination of phenyl alanine produces phenyl pyruvic acid, which can be detected colorimetrically Used in the characterization of the genera Proteus and Providencia

17) STARCH HYDROLYSIS presence of enzyme amylase identify typical starch hydrolyzers; such as Bacillus sps. 18) UREASE detects urease that splits urea into ammonia and carbon-dioxide distinguish Proteus, Providencia rettgeri and Klebsiella (+) Salmonella, Shigella and Escherichia(-)

Gram positive bacteria shape bacilli

cocci Growth in air (+) Catalase test (+)

(-) Anaerobic cocci (-) streptococcus

Gram positive aerobic cocci (+) catalase test Glucose fermentation (-) Micrococcus

(+) Staphyloccus

Coagulase test

(-)

Novobiocin resistance

(-) Other coagulase negative sps.

(+) S.Aureus

(+) S.saprophyticus

CLASSIFYING STREPTOCOCCUS Bile soluble or Optochin sensitive () Bile esculin (-) Hemolysis 6.5% NaCl ( + Enterococcus ) () Non- enterococci ( + ) (+) S. pneumoniae

Streptococcus grp D

alpha Viridans group

beta

CLASSIFYING STREPTOCOCCUS (contd.) eta hemolysis

Bacitracin sensitive

(-) Hippurate hydrolysis or cAMP (+)

(+)

Group A

Group B

() Other Lancefield groups

GRAM POSITIVE BACILLI Acid fast test (+) Mycobacterium Nocardia

(-) Endospore (-) Motility testing (+) Bacillus (-) (+) (-) Clostridium (+) Growth in air

Listeria monocytogenes

GRAM POSITIVE , NON-MOTILE BACILLI Growth in air Propionibacterium

(-)

(+) Corynebacterium Kurthia

Catalase

(+) Corynebacterium

(-)

Erysipelothrix Gardnella

IDENTIFYING GRAM NEGATIVE BACTERIA shape bacilli

Cocci (-) Growth in air (+) Neisseria (-) Growth on ThayerMartin Agar (+) Neisseria sps. Veillonela

IDENTIFICATION OF Gm (-) and aerobic NEISSERIA SPS. Positive growth on TheyerMartin agar

ONPG growth

(+)

N. lactamica

(-) Glucose and Maltose (acid) (+,+) N.meningitidis

(+,-) N. gonorrhoeae

Gram- negative bacilli Growth in air ( + oxidase ) (F) ( + GLUCOSE ) (-) (-) (F) GLUCOSE (O) Aeromonas lwoffi Enterobacteriac ea Aeromonas baumanii (N.R) (N.R) Alkaligenes, Eikenella Moraxella, Brucella Haemophilus Campylobacter

Aeromonas (O) Cardiobacteri Burkholderia a Achromobact Chryseoer bacterium Pasteurella vibrio

Gram negative anaerobic bacilli Kanamycin(1000g) (S) (R) Porphyromonas Prevotella Fusobacteriu m

Penicillin, 2U

(S)

Bacteroides sps.

(R)

B.fragilis