Professional Documents
Culture Documents
Safranin O = 30 secs
Gram (+) becomes (-) Over-decolorization
Old dying
Acidic iodine
Penicillin: omits iodine
Gram (-) becomes (+) Under-decolorization
Thick smear
Acid Fast staining methods Smear = 2 x 3cm
Pappenheim’s M. smegmatis vs. M. tuberculosis
Baumgarten’s M. leprae vs. M. tuberculosis
Fite Faraco M. leprae
Counterstain: Hematoxylin
Acid fast organisms Mycobacterium
Nocardia = Mod. AFS (1% H2SO4 as decolorizer)
Cryptosporidium
Legionella micdadei
Rhodococcus equi
Ziehl-Neelsen (Hot method) Best AFS
C-A-M
1. Carbolfuchsin = 1’ stain
-Start timing: Vapor (10mins)
-Heat = Mordant
2. 3% Acid alcohol = Decolorizer
-HCl + 95% etOH
-Until no more stain (Max: 3mins)
3. Methylene blue = counterstain
-30secs to 1min
Results:
AFO = Red
NAFO = Blue
Kinyoun (Cold method) Not used
C-A-M
1. Carbolfuchsin = 1’ stain
-Phenol, Tergitol = Mordant
2. 3% Acid alcohol = Decolorizer
3. Malachite Green = Counterstain
Results:
AFO = Red
NAFO = Green
Auramine-Rhodamine Most sensitive
(Fluorochrome) 1. Auramine-rhodamine = 1’stain
2. 0.5% Acid alcohol = Decolorizer
3. 0.5% KMnO4 = Counterstain
Results:
AFO = Yellow fluorescence
NAFO = No fluorescence
AFB Read 300 fields
Special stains Capsule = Negative stain
Spore = Dorner, Wirtz, Conklin
Metachromatic granules
- Albert’s
-Loeffler’s Alkaline Methylene Blue (LAMB)
lec.mt 04 |Page | 57
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
Flagella = Leifson
Nucleic acid = Feulgen
Polar bodies (ex: Y. pestis) = Wayson
Rickettsia = Gimenez
Spirochetes = Levaditi
Phase contrast microscope For study of living unstained organisms
Electron microscope For viruses
Light source: Electrons
100,000x magnification
Stains:
-Negative stain
-PTA
-Heavy metals (Gold, Silver)
Transmission EM DNA, RNA, chromosomes
Scanning EM Surface structures (cell wall, capsule)
Inverted Microscope For tissue culture
Interference microscope Dual light source
Non staining method String’s test (3% KOH)
Pure culture Streak plate = overlap method
Pour plate = Water and milk bacteriology
Selective medium
Animal inoculation = for virus, Chlamydia, Rickettsia
Mixed culture 2 or more organisms
Stock culture Stored at refrigeratior or freezer (long term)
Liquid Broth
Semi-solid 0.5-1% agar
Solid 2-3% agar
Biphasic Both liquid and solid
Ex. Castañeda = Brucella
General purpose media Nonfastidious organisms
1. Sheep BAP = Hemolysis
2. Horse BAP = Haemophilus
-Heat-stable, provides X-factor
3. Nutrient agar
Enriched media Solid
Fastidous organisms
1. CAP = Heat-labile, provides X & V factor
Enrichment media Liquid
1. Selenite F
2. Alkaline peptone water
3. Thioglycollate broth
Differential media 1. BAP = hemolysis
2. MAC
3. EMB
4. XLD
5. HEA
Selective media Inhibitory media
1. TCBS
2. SSA
3. TMA
4. CBAP
lec.mt 04 |Page | 58
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 59
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
BSC Class III Supply and exhaust air thru HEPA filter
Maximum protection
N95 Mask Contains HEPA filter
For Mycobacterium
Stool No direct exam in Microbiology
Transposons Resistant gene
Mobile or jumping
Fusiform F. nucleatum
Capnocytophaga
BSL I No risk
BSL II Moderate risk
BSL III High risk
Treatment available
Inhalation of aerosols
Ex. Mycobacteria (BSC Class II)
BSL IV High risk
No treatment available
Inhalation of aerosols
Ex. Small pox
Blood culture bottle BHIB + 0.25% SPS
Dilution = 1:10 (1mL blood, 9mL broth)
Anti-complementary, anticoagulant, antiphagocytic
Neutralizes aminoglycosides
Disadvantages of SPS Inhibits:
-Neisseria
-G. vaginalis
-S. moniliformis
-P. anaerobius
1% gelatin Counteract SPS to allow the growth of organisms
Indications of growth Hemolysis
(Blood culture) Turbidity
Pellicle
Subculture (Blood culture) BAP
MAC = no CO2
CAP
If blood culture = negative 7 days = Bacteremia (Typhoid)
21 days = Brucellosis, SBE
Urine culture Specimen: Catheterized, Midstream, Suprapubic
Quantitative: BAP, MAC
->100,000 CFU/mL (or >50,000 CFU/mL) = significant for UTI
-<10,000 CFU/mL = not significant (contaminants)
CSF culture DO NOT refrigerate
Agents: Neisseria, Haemophilus (Meningitis)
Media: BAP, MAC, CAP, BHI
C. neoformans:
-India ink method
-Latex agglutination
Wound specimen Gram stain
Media: BAP, MAC, Thioglycollate broth
Stool specimen Media: MAC, BAP+Ampicillin, CBAP, SSA, Selenite F, TCBS, APW, HEA
Oxidase test
Biochemical tests
lec.mt 04 |Page | 60
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
Serologic typing
Respiratory specimen Sputum, NPS
TB = 3 sputum specimen
Media: BAP, MAC, GBAP, BCAP, Amies,
Gram stain and Acid fast stain
Throat swab Sore throat
2 specimen
Media: BAP, MTM, Thioglycollate broth
Vaginal, Urethral swab Media: CAP, MTM
Gram stain
TB culture NALC-NaOH = Gold standard
-NALC = digestion, lyse the mucus
-NaOH = decontamination
6% Oxalic acid = Pseudomonas
Centrifuge (4’C) for 15 mins at 3000g
Media: LJ, Middlebrook 7H11, 7H10 (AST)
Incubate at 37’C for 8 weeks ----(NG)----> Report as (-)
If (+), after 2-3 weeks: growth is seen
GenPro Genetic Pro
DNA test
Result 2 hrs
Moist heat sterilization 1. Autoclave (sporicidal)
-121’C at 15 lbs/psi for 15 mins
-Culture media, bandages, gauze
-QC: B. stearothermophilus
2. Inspissation (sporicidal)
-75-80’C for 2 hrs on 3 days
-Disinfect and solidify protein containing medium (LJ, Loeffler’s)
-Water is heated from below and slanting surface gets heated
3. Tyndallization (sporicidal)
-100’C for 30mins on 3 days
4. Boiling (Nonsporicidal, disinfectant)
-100’C for 30mins
-Kills vegetative cells only
5. Pasteurization (Nonsporicidal, disinfectant)
-Milk
-63’C for 30mins
-72’C for 15secs
-Phosphatase: to determine if pasteurization is successful. (+): Not pasteurized
Dy heat sterilization 1. Hot air oven (Sporicidal)
-170-180’C for 2 hrs
-Glasswares, cottonswabs, metallic instruments, oils, powders
-QC: B. subtilis
2. Incineration (Sporicidal)
-Waste disposal
-Not recommended
3. Cremation (Sporicidal)
-Prevents communicable disease
4. Flaming (Sporicidal)
-Needles
5. Gas: Ethylene oxide (sporicidal)
-Heat-labile machine instruments
lec.mt 04 |Page | 61
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 63
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 64
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 65
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 67
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
2. Decontamination-Digestion = Best
-Na citrate: remove metallic compound
3. AFS
4. Culture
Pott’s disease Bone TB
Agar based media Duboi’s oleic acid albumin medium
Mitchison’s medium
Middlebrook 7H10 and 7H11 (AST)
Egg based media Petragnani medium
(Sterilization: Inspissation) Löwenstein Jensen (Slant) medium
American Thoracic Society medium
Dorset egg medium
Liquid media BACTEC 12B
Septi-Chek
Middlebrook 7H9
Niacin test Principle:
Niacin ---(enzyme)---> Niacin ribonucleotide + Cyanogen bromide + aniline dye
(No yellow color)
M. tuberculosis:
Niacin ---(no enzyme)---> Niacin + C.bromide + A.dye = (+) Yellow color
Heat stable catalase test Temp: 68’C
Medium: Tween 80
Rgt: 30% H2O2
Principle:
Tween 80 + Mycobacterium + 30% H2O2 ----(Heat at 68’C)----> (+) Gas bubbles
(+) M. kansasii
(-) M. tuberculosis
Nitrate reduction test Principle:
Na Nitrate ---(Nitroreductase)---> Nitrite
Nitrite + HCl + Sulfanilamide + N-1-naphthylethylenediamine --> (+) Red/Pink
Tween 80 hydrolysis test Tween 80: Polyoxyethylene sorbitan mono-oleate
Principle:
Tween 80 ---(Tween 80 lipase)---> (+) Oleic acid (Red)
(+) M. kansasii and
Differentiates M. gordonae (+) from (-) M. scrofulaceum
Arylsulfatase test For rapid growers
Principle:
Tripotassium + --------(Arylsulfatase)---------> (+) Free phenolphthalein (Red)
Phenolphthalein disulfide/sulfate
(+) M. fortuitum-chelonae
TCH Susceptibility test TCH: Thiopene-2-carboxylic acid hydrazide
(S): M. bovis
(R): M. tuberculosis
Old Skin Test (TB) 1. Mantoux = intracutaneous (most sensitive)
2. Von Pirquet = scratch on skin
3. Vollmer’s patch test = piece of cloth w/ PPD skin
4. Moro percutaneous test
5. Tuberculin time test = multiple puncture technique
PPD 0.1 mL (5 TU)
(+): Induration (≥10mm)
Doubtful: 5-<10mm/(-): <5mm
lec.mt 04 |Page | 69
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 70
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
b. M. chelonei
-(-) NO3 reduction
-(-) 5% NaCl
-(-) Iron uptake
2. M. smegmatis
3. M. phlei (“Hay bacillus”)
Growth: 10-21 days Groups I, II, III (Runyon’s Classification)
Growth: 3-7 days Group IV
(Rapid growers)
lec.mt 04 |Page | 72
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 73
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
Veillonella S R S
Clostridium S S R
Kanamycin Vancomycin Colistin
Gram (+) cocci S S R
Porphyromonas R S R
P. anaerobius R S R
Prevotella R R S
Brick red fluorescence Prevotella
Porphyromonas
Red fluorescence Veillonella
Pitting of agar B. ureolyticus
Swarming C. tetani
C. septicum
Molar tooth colony, sulfur A. israelii
granules
Breadcrumb colony Fusobacterium
Gram (+) Anaerobic Bacilli Actinomyces
Bifidobacterium
Eubacterium
Propionebacterium
Lactobacillus
Mobiluncus
Gram (-) Anaerobic Bacilli Bacteroides
Porphyromonas
Prevotella
Fusobacterium
Gram (+) Anaerobic Cocci Peptostreptococcus
Peptococcus
Gram (-) Anaerobic Cocci Veillonella
Megasphera
Acidaaminococcus
A. bovis Lumpy jaw
P. acnes Acne vulgaris
(+) Catalase, Indole
L. acidophilus Tomato juice agar
(-) Catalase
Mobiluncus Vaginitis (G. vaginalis)
(-) Catalase
Anaerobic infections Foul odor
B. fragilis Needs 20% bile
F. necrophorum Vincent’s angina (B. vincenti)
(F. moriferum)
P. anaerobius SPS sensitive (Neut: 1% gelatin)
(-) Indole, Catalase
P. asaccharolyticus (-) Catalase
P. niger Staphylococcus-like
Black
H2S indicators TSI = Ferrous sulfate
LIA = Ferric ammonium citrate
SIM = Lead acetate
Phenol red Acid = Yellow
Alkaline = Red
lec.mt 04 |Page | 74
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
K. oxytoca
E. gergoviae
Y. pseudotuberculosis
Y. enterocolitica
Aerogenic Klebsiella
E. coli
Salmonella (except S. typhi, S. gallinarum)
Proteus
Arizona
Citrobacter
Enterobacter
Enteric Media
Inhibitory CHO Indicator LF NLF
EMB Eosin Y Lactose Eosin Y Red purple Colorless
Methylene blue Methylene blue (E. coli: GMS)
MAC Bile salts Lactose Neutral red Red/pink Colorless
Crystal violet
SSA Bile salts Lactose Neutral red Red Colorless
DCA Bile salts Lactose Neutral red Red/pink Colorless
TCBS Bile salts Sucrose Bromthymol blue Yellow Green
lec.mt 04 |Page | 77
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
Complication: Psychosis
Lab Diagnosis:
-1st week: Blood
-2nd week: Urine and stool (carrier)
S. paratyphi C (S. choleraesuis) Septicemia
S. enteritidis “Gardner’s Bacillus”
Shigella Biochemically inert
SSA: colorless
Related to E. coli:
-E. coli: Acetate (+)
-Shigella: Acetate (-)
Intestinal pathogen: Dysentery
Lab. Diagnosis:
-BEST: Culture of fresh stool w/ mucous flecks
-Rectal swab of ulcer
Shigella Subgroups (O Ag) A = S. dysenteriae (“Shiga Bacillus”)
B = S. flexneri (“Strong Bacillus”)
C = S. boydii
D = S. sonnei (Cross reacts w Plesiomonas)
Serratia marcescens Red pigment (Prodigiosin)
(+) DNase, Gelatinase, LPS
Red milk
Resistant to many antibiotics
Serratia odorifera Rancid, potato-like odor
Y. pestis “Plague Bacillus”
(+) Stalactite on broth medium
V and W antigens
MOT: Rat flea bite
Infections:
-Bubonic plague: buboes, lymphadenopathy
-Pneumonic plague
-Septicemic plague
-Black death
Y. enterocolitica Motile at RT’
Appendicitis-like infection
(+) ONPG
Y. pseudotuberculosis Motile at RT’
Mesenteric lymphadenitis, septicemia
Vibrio “Comma Bacillus”
Comma-shaped
Darting motility
Oxidase (+) except V. mitschnikovii (-)
Halophilic except:
-V. cholerae
-V. mimicus
V. cholerae (+) Indole
(+) String test (0.5% Na desoxycholate)
Cholera: Ricewater stool
“Fish in the stream” appearance
Classical Biotype El Tor Biotype
Polymyxin Susceptibility S R
lec.mt 04 |Page | 78
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
Lysis by bacteriophage + -
Chicken RBC agglutination - +
Classical Biotype El Tor Biotype
Hemolysis of sRBC - +
VP test - +
Ogawa Serotype Inaba Serotype Hikojima Serotype
Anti-Ogawa + - +
Anti-Inaba - + +
Lab. Diagnosis (V. cholerae) Culture:
-TCBS: Sucrose fermenter
-TTGA: Tellurite Taurocholate Gelatin Agar
-APW: 6-8 hrs Subculture (TCBS)
-Cary Blair: Transport medium (stool)
-Rectal swab: Carrier
O129 Susceptibility test = (S)
V. parahaemolyticus Halophilic (8% NaCl)
(+) Indole
#1 Gastroenteritis in Japan (seafood)
Nonsucrose fermenter (TCBS)
Kanagawa (+) = Beta hemolysis on Wagatsuma agar
V. vulnificus Stool pathogen
(+) Blood culture
(+) Lactose fermentation, ONPG
Nonsucrose fermenter (TCBS)
V. alginolyticus Sucrose fermenter (TCBS)
Aeromonas Motile (monotrichous)
(+) Oxidase
Beta-hemolytic
(R) O129
(+) DNase
(+) Bile esculin hydrolysis
Plesiomonas (+) Oxidase
Non-hemolytic
(V) O129
(-) DNase
(-) Bile esculin hydrolysis
Campylobacter (G) 42’C
Curved, S-shaped (Wings of Seagull)
(+) Catalase
(+) Oxidase
Darting motility
C. jejuni: (+) Hippurate
H. pylori (+) Catalase
(+) Oxidase
(+) Urease
Test: Urease breath test
OF test Fermentative (close) = O/F = +/+ = Y/Y
Oxidative (open) = O/F = +/- = Y/G
Nonutilizer = O/F = -/- = G/G
Medium: Hugh and Leifson (1% glucose, 1% agar)
Indicator: Bromthymol blue
P. aeruginosa Old: P. pyocyanea
lec.mt 04 |Page | 79
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 80
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 81
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 82
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 83
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 84
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 85
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 86
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
lec.mt 04 |Page | 87
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)
lOMoARcPSD|5746394
Catalase + -
Motility (25’C) + -
Hemolysis Beta Alpha
L. monocytogenes E. rhusiopathiae
VP + -
H2 S - +
Bile esculin + -
Hippurate + -
Gluconate + -
Media Mc Bride, Cold enrichment BAP
Water Bacteriology E. coli: Index of fecal contamination
Det. coliforms
Sodium thiosulfate Neutralizes chlorine
1. Presumptive test Lactose broth/ + H2O ----------(35’C for 24 hrs)----> (+) Gas
Lauryl tryptose broth (-) No gas after 48 hrs
2. Confirmatory test EMB/Endo agar + Inoculum -----(24 hrs)-----------> (+) Colony
3. Completed test Lactose broth fermentation tube ---(35’C for 24-48 hrs)---> (+) Acid + Gas
MPN (Most Probable Number) Most common method for H2 analysis
Estimation of coliform group density
# of positive coliforms from multiple decimal dilution
Reference laboratories EAMC
(Water Bacteriology) FDA
Milk Bacteriology
Streptococcus lactis Acid forming, causes normal souring of milk
Coliform bacteria Gas-forming, hydrolyze the milk protein casein
Bacillus subtilis Hay bacteria, proteolytic action on coagulated milk
Clostridium butyricum Stormy fermentation of milk
Clostridium perfringens
Pseudomonas syncyanea Blue milk
Serratia marcescens Red milk
Pseudomonas aeruginosa Blue green milk
Flavobacterium synxanthum Yellow milk
Alcaligenes viscosus Slimy or ropy milk (capsule formation)
lec.mt 04 |Page | 88
Downloaded by Olga Mare Barredo (olgamarebarredo@gmail.com)