DIAGNOSTIC LABORATORY TEST

Jawetz, Henrys, Bailey & Scotts, Zissner, Microbiology made easy

Gram Positive Cocci

1. Staphylococcus aureus (Golden Staph / Oro Staphira)

Culture
o BAP = large, creamy, opaque colonies, usually Beta-hemolytic w/ golden yellow pigments
o CNA
o MSA = golden yellow
o PEA
Biochemical
o Catalase (+)
o Coagulase (+)
Serotyping/Others
o Commercial latex agglutination product
o FISH product
o PCR assay

Test (Staph in General)

o Penicillin G (R)
o 0.0 Bacitracin (R)
o 200 ug/mL Lysostaphin (S)
o Modified Oxidase (-)

2. Staphylococcus epidermidis
a normal flora of the skin and frequent contaminant of blood cultures
colonizes prosthetic devices such as catheters, prosthetic heart valves and joints
Culture
o BAP = gray-white colonies on primary isolation
Biochemical
o Catalase (+)
o Coagulase (-)
Antibiotic susceptibility Test
o Novobiocin (S)

3. Staphylococcus saprophyticus
2nd most common cause of UTIs next to E. coli
Culture
o BAP: Gamma hemolytic
Biochemical
o Catalase (+)
o Coagulase (-)
Antibiotic susceptibility Test
o Novobiocion (R)

4. Streptococcus pyogenes
Prototype human pathogen
Called as pyogenes as it is pus-producing
Culture
o BAP = Beta-hemolytic, discoid, matte or glossy colonies; transparent to translucent, convex,
entire, circular, shiny and are surrounded by a rather wide zone of beta hemolysis (10% CO2)
o PEA
Biochemical
o PYR Test = bright cherry red
Serological Test
o Antistreptolysin O (Respiratory disease)
o Anti-DNase
o Antihyaluronidase (Skin infection)
o Antistreptokinase
o Anti-M type-specidic antibodies
Scarlet Fever Susceptibility Test
o Dicks Test = Redness in test arm
o Schultz-Charlton Test = Blanching phenomenon (Fading of scarlatinal rash)
Serotyping/Others
o Latex agglutination
o Nucleic acid probe (throat swabs)
 5. Streptococcus agalactiae
Culture
o BAP = larger, more translucent to opaque, whitish gray, soft, smooth colonies surrounded by a
much smaller zone of beta hemolysis
o Lim broth
o Carrot broth = yellow or orange
o Todd Hewith broth
o Trans Vag broth w/ 5% sheep blood
o Granada agar = orange colonies
o CAMP Test in BAP = arrowhead formation of hemolysis around Sau
Biochemical
o Hydrolysis of Sodium hippurate = deep purple
Serotyping/Others
o Latex agglutination
o Coagglutination Test
o Chemiluminescent DNA probe
o Smart Cycler Assay

6. Group D
Enterococcal group
Bile esculin hydrolysis = dark brown
Bile esculin agar = blackening of the agar slant
6.5% NaCl = with growth
Non-enterococcal group
Bile esculin hydrolysis = no reaction
Bile esculin agar = no blackening
6.5% NaCl = without growth

7. Streptococcus pneumoniae (Pneumococcus)

Microscopy
o Gram Stain: lancet-shaped or bullet-shaped diplococci
Culture
o BAP = Mucoid colonies, dome-shaped w/c develops to a crater-like appearance central
depression with an elevated rim; distinctive, with very mucoid and glistening colonies that tent to
dip down in the center and resemble a doughnut (5-10% CO2)
Optochin Test = 14mm zone of inhibition
Bile solubility Test = Autolysis (Clear solution)
Quellung Test (Newfeldt Reaction)= swollen capsule
Serotyping/Others
o Urinary antigen assay
o Phadebact Test (Coagglutination Tets)

8. Viridans group
BAP: alpha hemolysis on BAP. (greenish discoloration)
Caramel odor: Milleri streptococci (VP+)

Gram Negative Cocci

9. Neisseria
Microscopy = Gram (-) diplococci that are kidney-shaped or coffee-bean shape except Neisseria
elongata as it is rod-shaped
Culture
o Enriched media(MTM, ML, GC-Lect, NYC, CAP) ~ 3-10% CO2 @ 37C
Gonococci and Meningococci: convex, glistening, elevated mucoid colonies about 1-5mm
in diameter and may be transparent or opaque, nonpigmented and non-hemolytic
Neisseria flavescens, N. cinerea, N. subflava and N. Lactamica: yellow pigmentation
Neisseria sicca: opaque, brittle, wrinkled colonies
Moraxella cattarhalis: nonpigmented or pinkish gray opaque colonies
Biochemical
o Oxidase Test = dark purple; rapid test (filter paper soaked w/ tetramethylparaphenylene
diamine dihydrochloride)
o Cystine Trypticase Acid base medium (24-72 hrs)
10. Neisseria gonorrhoeae (Gonococci)
Microscopy
o Gram stain = diplococci within pus cells (urethral or endocervical exudates); small doughnut
Culture
o MTM (5% CO2 candle extinction jar at 37C
o MALDI-TOF
Subculture
o Selective: small, piliated
o Nonselevtive: larger, nonpiliated
o T1 & T2 colonies: small, bright, reflective, raised (T1: moist; T2: dry)
o T3 & T4: larger, flatter, nonreflective
o T5: develops after in-vitro
Biochemical
o Weakly catalase (+)
o Oxidase (+)
o Carbohydrate oxidative reaction: Acid formed from glucose
Serotyping/Others
o Coagglutination
o IF staining
o Immunoblotting
o Radioimmunoassay
o ELISA
o NAAT (endocervical or urethral swab)
o Phadebact Monoclonal GC Test
o GonoGen I
o GonoGen II
o Chemiluminescent nucleic acid probe (confirmatory)
Transport media
o Transgrow
o JEMBEC (John E. Martin Biological Environmental System)

11. Neisseria meningitidis (Meningococci)

Microscopy
o Gram stain = diplococci within pus cells or extracellularly
Culture
o Without SPS
o CSF: CAP (5% CO2 @ 37C)
o MTM
o Spinal fluid: can be directly incubated

Biochemical:
o Carbohydrate oxidative reaction: Acid formed from glucose, and maltose
Serotyping/Others
o Agglutination
o Hemmagglutination
o Slide agglutination (Confirmatory test)
o Latex agglutination (CSF, serum, and urine)
Petechial rash (small hemorrhages) is the hallmark of Meningococcal Infections

12. Moraxella catarrhalis

DNAse (+)
DOES NOT OXIDIZE ANY SUGARS
Gram Positive Spore-Forming Bacilli

13. Bacillus anthracis (bamboo, medusa head, cut glass)

Microscopy
o Gram stain = bamboo appearance; large, boxcar-shaped, gram positive; Chinese bamboo
fishing rod appearance; Central or Subterminal endospore
o Fluorescent-labeled antibody test
o India ink
o Hanging drop method: nonmotile
o MHA w/ Penicillin: strings of pearl formation in microscopic examination
Culture (35C)
o BAP: non-hemolytic, flat, opaque, grayish-white, tenacious colonies with rough texture and a
cut glass or ground glass appearance (in transmitted light); irregular, fringe-like margin or a
comma-shaped outgrowth (Medusa head/Lion head/ curled hair)
o B Lysozyme EDTA Thallous acetate Agar: has the consistency of beaten egg whites
when touched with an inoculating loop as the colonies are tenacious
o Gelatin: Inverted pine/fig tree; slow liquefaction
o Bicarbonate-Containing Medium: mucoid colonies
Biochemical
o Salicin Fermentation (-)
Antibiotic susceptibility Test
o Penicillin (S)
Serotyping/Others
o Ascoli Test
o Gamma bacteriophage (S)
o IHA
o ELISA
o PCR

14. Bacillus cereus

Microscopy
o Hanging drop method: motile
Culture
o BAP: beta-hemolytic, small, shiny, compact or large feathery and spreading, gray to lavender
Biochemical
o Salicin Fermentation (+)
Antibiotic susceptibility Test
o Penicillin (R)
Serotyping/Others
o Gamma bacteriophage (R)

15. Bacillus subtilis (Hay Bacillus or Grass Bacillus)

Guthrie inhibition test

16. Clostridium botulinum

Microscopy
o Motile: Petrichous flagella
Culture
o BAP: all strains except type G is Beta hemolytic
o Alkaline glucose gelatin: heat-resistant spores
Serotyping/Others
o Mouse bioassay (Confirmatory)
o ELISA
o PCR
o Passive hemagglutination
o RIA

17. Clostridium tetani (Tack head bacilli)

Microscopy
o Terminal spore
o Drumstick or Tennis racket appearance
o Motile: Petrichous flagella
Culture
o BAP: swarming w/ delicate flat edge of projecting filaments; Faint B-hemolytic
Biochemical
o DOES NOT FERMENT ANY CARBOHYDRATE

18. Clostridium perfringens (Gas gangrene Bacillus)

Microscopy
o Non motile
o Short, plump, encapsulated; Box-car rods
Culture
 oBAP: circular, smooth, large, white colonies; exhibits a double zone of beta hemolysis (inner
zone of hemolysis is due to theta toxin while outer zone is due to alpha-toxin)
o Chopped-meat glucose: abundant growth; gas (+)
o Thioglycolate medium
o No spores in ordinary media
Biochemical
o Milk media: Stormy fermentation; Tear/crack in the curd (Confirmatory)
o Lecithinase Test (Naeglers Test): opaque halo precipitate w/c is yellow in color surrounding
bacterial colony

19. Clostridium difficile

Microscopy
o Motile: Petrichous flagella
o Non encapsulated
Culture
o Cycloserine Cefoxitin Fructose Agar: yellow, ground glass appearing colonies
Biochemical:
o Gelatinase (+)
Serotyping/Others
o Rapid Latex Agglutination test
o Toxin A detection

20. Corynebacterium diphteriae (Klebs-Loeffler Bacillus)

Microscopy
o Gram Staining: slightly curved, gram positive rods with nonparallel sides, and sometimes wider
ends, giving a clubbed appearance; club-shaped; Palisade, Chinese-character, Picket Fence
appearance; snapping movement when two cells divide. (V and L formations)
o Aniline dye: Granules (Volutin granules or Babes-Ernst Granules) are irregularly distributed
within the rod giving it a beaded appearance
o Individual corynebacteria: lie parallel or at acute angles to one another
Culture (37C in 5% CO2)
o BAP: small, granular, and gray with regular edges and may have small zone of hemolysis
o Cysteine-Tellurite BloodAgar: brown to black with a brown-black halo (48 hrs)
o Modified Tinsdales medium
o Pais coagulated egg medium: for pleomorphism and granule production
o Loefflers coagulated serum slant (12 hours followed by staining with methylene blue): rod-
shaped pleomorphic; minute, grayish white, glistening
o CNA: If CT or Tinsdale is not available
o Thiogylcolate
o BHI
Biochemical
o Catalase (+)
Serotyping/Others
o Elek plate gel diffusion test: white precipitin band appears at 45 between filter paper and
streak
o ELISA
o PCR: detect diphtheria toxic gene
o Immunochromatographic strip assay
Transport media
o Amies

C. jeikum: metallic sheen on BAP

var gravis: matte; only non-hemolytic var
var mitis: glossy and smooth; exhibits fried egg appearance and has a bleach-like odor in any
medium using tellurite
var intermedius: shiny
var belfanti: only nitrate (-) var

21. Listeria monocytogenes

Microscopy
o Saline suspension: Tumbling motility at room temp but rarely at 35C
Culture
o Optimal growth: 30C 37C; however growth may occur as low as 4C
o BAP: small, transluscent, grayish blue, narrow zone of beta hemolysis
o Trypticase agar or NA: smooth, transluscent, blue-green
o Tryptose agar
o PEA agar
o McBride agar: crushed glass appearance
o Nalidixic acid media
Biochemical
o Semi-solid media: Umbrella shape at the top of the medium
o Catalase (+)
 o Ferments glucose, trihaloce, salicin
o Esculin hydrolis (+)
o CHO fermentation w/ acid, no gas
Antibiotic susceptibility Test
o Neomycin (S)

22. Erysipelothrix rhusiopathiae

Microscopy
o Hanging drop method: motile
Culture
o Best specimen: Biopsy and tissue aspirates from erysipeloid lesions
o BAP (7days): nonhemolytic, greenish discoloration of media (2 days)
o CAP:
o Gelatin stab culture: Pipe cleaner at 22C; Test tube appearance
o Tellurite media: Black
o BHI
Biochemical
o Oxidase (-)
o Catalase (-)
o TSI: H2S (+)
Antibiotic susceptibility Test
o Neomycin (R)

23. Nocardia (Nocardia asteroids)

Microscopy
o Gram Stain: rods, coccobacillary form or tangled masses of branching rods; weakly gram-
positive, partially acid-fast beaded branching thin filaments
o Growth on milk enhances acid fastness
Culture (10% CO2)
o BAP
o CAP
o Potato dextrose agar
o Sabourauds dextrose agar
o Lowenstein-Jensen
o Middle brook media
o 7H9 broth
o BCYE
o Waxy, yellow to orange colonies with aerial hyphae
Biochemical
o Catalase (+)
o Urease (+)
Serotyping/Others
o rDNA sequencing
o PCR-restriction enzyme pattern analysis

24. Actinomyces (Actinomyces israelii and A. gerencseriae)

Microscopy
o Gram Stain: gram-positive, beaded, filamentous anaerobic; fungus-like branched networks of
aerial hyphae; rod, coccoid or coryneform
o Pus: yellow granules
Culture (37C)
o BHI & Thio: Filaments break into short chains and coccobacillary forms (24-48 hrs); molar
tooth

25. Propionibacterium
Microscopy
o Irregular rods, branching
Culture
o BAP: 1-2mm in diameter, circular, entire, convex, glistening, opaque; some produces a narrow
zone of hemolysis
Biochemical
o Converts lactic acid to propionic acid and CO2