Presentation 1 - Ex 6

EXERCISE 6 • To recognize and describe the various growth patterns of bacteria on different media preparations • To perform and interpret the results of various staining techniques to morphologically characterize bacteria • To present the principles, strengths, limitations and alternatives of the various staining techniques

EXERCISE 6 • To demonstrate the presence of cellular structures through staining • To perform and interpret various chemical tests • To characterize and identify unknown microorganisms base on its CULTURAL, MORPHOLOGICAL and PHYSIOLOGICAL characteristics

THE REFERENCES

CULTURAL CHARACTERIZATION

PLATE CHARACTERIZATION

PLATE CHARACTERIZATION • FORM • ELEVATION • MARGIN • SURFACE • OPTICAL CHARACTERISTICS

FORM • PUNCTIFORM – UNDER 1mm IN DIAMETER

• CIRCULAR • FILAMENTOUS – LONG, IRREGULAR, INTERWOVEN THREADS

• RHIZOID – IRREGULAR, BRANCHED

• IRREGULAR

ELEVATION • EFFUSE – VERY THIN, SPREADING

• FLAT • RAISED • CONVEX • PULVINATE • UMBONATE

MARGIN • ENTIRE – SMOOTH, NO NOTCHES

• UNDULATE – WAVY, SHALLOW INDENTATIONS

• EROSE – IRREGULARLY NOTCHED

• FILAMENTOUS – IRREGULAR, INTERWOVEN EDGES

• CURLED – PARALLEL CHAINS IN WAVY STRANDS

SURFACE • SMOOTH • CONTOURED – IRREGULAR, UNDULATING

• RADIATE – RIDGES RADIATING FROM CENTER

• CONCENTRIC – RINGS WITH COMMON CENTER

• RUGOSE – WRINKLED

OPTICAL CHARACTERISTICS • OPAQUE – NOT PERMITTING LIGHT TO PASS THROUGH

• TRANSLUSCENT – SOME LIGHT PASSES THROUGH BUT NOT ENOUGH TO PERMIT VISIBILITY

• IRIDISCENT – OPAL-LIKE APPEARANCE – MILKY IRIDESCENSE

• DULL – NOT GLOSSY

• GLISTENING – GLOSSY

SLANTS CHARACTERIZATION

SLANT CHARACTERIZATION • AMOUNT OF GROWTH – SCANTY – MODERATE – ABUNDANT

• CONSISTENCY – BUTYROUS (BUTTER-LIKE) – VIRSCOUS (FOLLOWS NEEDLE WHEN DRAWN) – MEMBRANOUS (THIN, LIKE A MEMBRANE) – BRITTLE (DRY, FRIABLE)

• PIGMENTATION – NONE OR PIGMENTED

BROTH CHARACTERIZATION • AMOUNT OF • SURFACE GROWTH GROWTH – NONE – RING – PELLICLE

• SUBSURFACE GROWTH – TURBID (CLOUDY) – GRANULAR (SMALL GRANULES)

– SCANTY – MODERATE – ABUNDANT

• SEDIMENT – – – –

NONE GRANULAR VISCOUS FLAKY

MORPHOLOGICAL CHARACTERIZATION

PREPARING YOUR SMEAR FOR STAINING

STUDY OF THE BACTERIAL CELL WALL • GRAM STAINING • GREGERSEN’S METHOD • ACID FAST STAINING

GRAM STAINING • IMPORTANCE – PURE CULTURE CONFIRMED – SHAPE, ARRANGEMENT OF CELLS DETERMINED – DIVIDES THE BIG MICROBIAL WORLD INTO 2

SOME POINTS TO PONDER… • CAN WE CHANGE THE PRIMARY STAIN IN THE GRAM STAINING? • HOW ABOUT THE COUNTERSTAIN?

GREGERSEN’S METHOD • FAST FORWARD WAY OF CHECKING FOR CELL WALL CHARACTERISTICS • BASED ON REACTION BETWEEN KOH AND LIPIDS OF WALLS • GRAM NEGATIVE = VISCOUS AND STICKY • MAY NOT BE THAT ACCURATE

ACID FAST STAINING • FOR WALLS RICH IN MYCOLIC ACIDS • MYCLIC ACID RETAINS THE RED STAIN • RED-STAINED CELLS • IMPORTANT DIAGNOSTIC TEST FOR MYCOBACTERIUM SPECIES

STUDY OF THE BACTERIAL CAPSULE • CAPSULES ASSOCIATED WITH VIRULENCE • MAJOR LINE OF DEFENSE AGAINST PHAGOCYTOSIS • RESERVED MATERIALS (PROLONGED INCUBATION CONSEQUENCE) • FORMATION INFLUENCED BY NUTRIENT AVAILABLE • METHODS VARY IN ACCURACY • Pseudomonas and Bacillus

STUDY OF THE FLAGELLA • MORDANT = ENLARGES THE STRUCTURE FOR EASE OF VIEWING • FRESHLY-PREPARED STAINS • TAKE NOTE – PRESENCE – NUMBER – POSITION

SOME POINTS TO PONDER… • WHY INCUBATE BELOW OPTIMUM CONDITIONS? • DIFFERENCE BETWEEN TRUE MOTILITY AND BROWNIAN MOVEMENT? • WHY NEED TO CHECK FOR HANGING DROP AND MOTILITY BAND BEFORE FLAGELLAR STAINING?

BACTERIAL ENDOSPORE • STRUCTURE PRODUCED UNDER ADVERSE CONDITION • RESISTANT TO EXTREME ENVIRONMENTAL PRESURES • TAKE NOTE: – – – –

PRESENCE NUMBER POSITION DISTENDING OF VEGETATIVE CELL

P O R O G E N E S I

SOME POINTS TO PONDER… • OTHER GENERA PRODUCING ENDOPORES? • SPORE ACTIVATION?

PHYSIOLOGICAL CHARACTERIZATION

OXYGEN REQUIREMENT • GROWTH IN ANAEROBIC MEDIA • CATALASE TEST • OXIDASE TEST

ANAEROBIC MEDIA • THIOGLYCOLLATE ABSORBS OXYGEN GAS • (UNAVAILABLE FOR BACTERIA)

CATALASE • ATTACKS H2O2 • PORPHYRIN-CONTAINING PROTEINS • 2 H 2O 2 →

2 H 2O + O 2

• AEROBES AND FACULTATIVE

OXIDASE • DETERMINES IF ORGANISM HAS CYTOCHROME C OXIDASE AS A RESPIRATORY ENZYME • REACTIONS DURING OXIDATIVE PHOSPHORYLATION • 2 REDUCED CYTOCHROME C + 2H+ + ½ O2 → 2 OXIDIZED CYTOCHROME C + H2O • TETRAMETHYL-ρ-PHENEYLENE DIAMINE DIHCl AS REAGENT • INDOPHENOL BLUE AS PRODUCT

CARBOHYDRATE METABOLISM

MRVP TEST • METHYL RED TEST (MIXED ACID FERMENTATION) – MIXED ACIDS FROM GLUCOSE METABOLISM – METHYL RED INDICATOR

• VP TEST (VOGES PROSKAUER TEST) – ACETOIN PRODUCTION – NEUTRAL PRODUCTS FROM GLUCOSE – VP REAGENTS 1 AND 2

METHYL RED TEST

VP TEST • ACETOIN + αNAPHTHOL + KOH + O2 → DIACETYL + GUANIDINE GROUP • UPON CONDENSATION GIVES A PINK PRODUCT

CITRIC ACID UTILIZATION • CITRATE ONLY CARBON SOURCE IN THE MEDIA • MICROBES HAVE BOTH PERMEASE AND CITRITASE • PRODUCT IS SODIUM CARBONATE AN ALKALI

STARCH HYDROLYSIS • ABILITY OF BACTERIA TO HYDROLYZE STARCH • AREAS OF STARCH HYDROLYSIS WILL APPEAR CLEAR UPON ADDITION OF IODINE • AMYLASE

DECOMPOSITION OF LARGE MOLECULES

TWEEN 80 HYDROLYSIS

GELATIN HYDROLYSIS • GELATINASE • GELATIN LIQUEFACTION

CASEIN HYDROLYSIS • HYDROLYZE CASEIN, MILK PROTEIN • GIVES WHITE COLOR • DECOLORIZED WHEN CASEN HYDROLYZED

NITROGEN AND SULFUR METABOLISM

UREA HYDROLYSIS • UREA + 2H2O → NH3 + CO2 + H2O

2

• PROTEUS • MAKES THE MEDIA ALKALINE DUE TO AMMONIA

INDOLE PRODUCTION FROM TRYPTOPHAN • TRYPTONE IS OBTAINED FROM THE HYDROLYSIS OF MILK CASEIN • RICH IN TRYPTOPHAN • REACTIONS INVOLVED: – TRYPTOPHAN + ½ O2 → INDOLE + PYRUVATE • TRYPTOPHANASE – 2 INDOLE + KOVAC’S REAGENT → ROSINDOLE DYE + H 2O • BRIGHT RED RING

PHENYLALANINE DEAMINATION • REMOVAL OF AN AMINO GROUP (DEAMINATION) • PHENYLALANINE + ½ O2 → PHENYLPYRUVIC ACID + AMMONIA • PHENYLPYRUVIC ACID + FERRIC ION → GREEN FERRIC

LEAD SULFIDE/HYDROGEN SULFIDE PRODUCTION • (1) GROWTH, NO FERMENTATION, NO H2S • (2) H2S, ACID, GAS • (3) H2S, GAS • (4) ACID, GAS • (5) UNINOCULATED

LEAD SULFIDE/HYDROGEN SULFIDE PRODUCTION • SOME BACTERIA CAN LIBERATE HYDROGEN SULFIDE FROM CYSTINE, CYSTEINE AND METHIONINE • HEAVY METAL SALT ADDED TO THE MEDIA (Fe3+ OR Pb2+) • HYDROGEN SULFIDE REACTS WITH METAL TO PRODUCE A BLACK PPT (FeS OR PbS) • CYSTEINE DESULFHYDRASE

ADVANCES IN MICROBIAL IDENTIFICATION/ DETECTION