CAPSULES: Gram (-) Susceptible to antibiotics

Polysaccharide Capsule S. pneumoniae, K.pneumoniae, N.meningitidis Balance between living and dead cells
PLATEU/Stationary
Polypeptide-D-glutamic Acid B. anthracis (+) sporulation
Hyaluronic Acid Pasteurella multocida Complete cessation
Polyribosyl Ribitol Phosphate H. influenza Increased death rate
P. aeruginosa ; Growth is stopped
Alginate Capsule Cause of death:
Alginate → enhance formation of BIOFILMS DEATH/Decline
MOTILITY • Food shortage
Tumbling Listeria monocytogenes • Increase con’c of toxic products
Twitching Kingella kingae • Development of unfavorable pH
Gliding/Sliding Capnocytophaga gingivalis (no flagella) BIOSAFETY LEVELS
Darting Campylobacter spp. LEVEL Description Precautions Examples
Shooting star Vibrio cholerae BSL-1 Minimal risk Practice Standard Laboratory B. subtilis
Corkscrew Spirochetes Contaminants Techniques M. gordonae
INCLUSION BODIES BSL-2 Moderate risk BSL-1 precautions + Salmonella
Babes Ernst Corynebacterium diphtheriae Shigella
Bodies/Volutin Ingestion, mucous Use of BSC, gowns, gloves, face S. aureus
MUCH granules MTB membrane, percutaneous shield (if needed) HBV
Halberstaedter Chlamydia trachomatis exposure HIV
Prowazek Demonstrated with Gimenez, Macchiavelo, and Castaneda Biohazard warning signs
Laboratory-acquired
BIOSAFETY CABINETS infections Decontaminate all infectious
CLASS 1 Negative pressure, ventilated cabinet; least effective waste
Unsterilized air enters and circulates BSL-3 High risk Sustainable airflow Systemic
ONLY Exhausted air is HEPA-filtered mycoses
System is ENTIRELY OPEN Inhalation, aerosol Use of PPE
CLASS 2 Aka. Laminar Flow transmission MTB
Entered, circulating (over infectious material) and exhausted air are HEPA- Coxiella burnetiid
filtered St. Louis
• IIa → 70% air is recirculated; Fixed-opening; air discharged INSIDE room encephalitis virus
• IIb → Variable-sash opening; air discharged OUTSIDE room BSL-4 Extreme Risk Separate building Marburg
CLASS 3 ENTIRELY CLOSED; most effective and expensive Life threatening; high Use of PPE (whole body) Arenavirus
Gloves attached and sealed to the cabinet mortality EBOLA
STAGES OF BACTERIAL GROWTH
No increase in number
LAG/Adaptation/Rejuvenescence Increase in size
No cell division yet
Increase in number
LOG/Exponential
Start of cell division
STERILIZATION Chemical Methods
Physical Methods - Ethylene Oxide
Autoclaving o most commonly used chemical sterilant;
- Steam under pressure o used for machines that cannot be autoclaved
o 121-15, 15 mins→ sterilize used and unused media o Indicator: B. subtilis var. globijii
Biocides
o 121-15, 30 mins → contaminated biologic materials - Cold Sterilization: Glutaraldehyde and Peracetic Acid
o 132-15, 30-60 mins → infectious medical wastes - Sterilization of HEPA filters in BSCs:
- Indicator: Bacillus stearothermophilus o Formaldehyde vapor
- PRIONS not killed o Vapor phase hydrogen peroxide
DISINFECTION
Fractional/Intermittent Sterilization Physical Methods
- Tyndallization Non-Ionizing - UV Exposure
Moist Heat
o 100 degC for 30mins for 3 days Radiation
[ATI]
o Arnold sterilizer, using flow steam Boiling - 100 degC for 15-30 mins
- Inspissation - Removal of pathogens in FOOD
o Sterilizing media with increased protein (ex. - 63 degC, 30 mins
Lowenstein Jensen) o Low Temperature Holding
o 75-80degC for 2 hours for 3 days o Batch or VAT Pasteurization
o Thickening thru evaporation Pasteurization
- 72 degC 15 secs
- Killing time: o High temperature short time or Flash method
o 1st day → Vegetative cells (active cells) - 140 degC 3 secs
o 2nd day → Spores o Ultra High Temperature short time
o 3rd day → remaining cells Chemical Methods
Flaming Oven Cremation Incineration - Most commonly used: 70% ISOPROPANOL
For loops 160-180 degC Burning body 870-980 degC - Forms of Iodine:
Dry
and needles 1-2 hrs to ashes Eliminate Prions o Tincture: Iodine + Alcohol
[FOCI]
B. subtilis var. Burning materials Antiseptics o Iodophor : Iodine + Detergent/ Neutral polymer
niger (for skin) - Best antiseptic: IODOPHOR
For materials not allowed to HEAT; Heat-labile, Sensitive - Cleansing of wounds: 10% Hydrogen Peroxide
- Membrane filters - 70% Isopropanol is better than 95% Isopropanol
Filtration
o Millipore filter → 0.22u; 100% sterility - Non-sporicidal (will not kill spores)
o HEPA filter → 0.3u - Best disinfectant for Blood spillage: Sodium Hypochlorite
o Depth filter (Household bleach)
- For disposable materials o Blood spills and porous surface: 1:10 dilution
- Use of Gamma Rays Disinfectants o Smooth and hard: 1:100
Ionizing - Indicator: B. pumilus (for surfaces) o HBV inactivation → 10 mins
Radiation o HIV inactivation → 2 mins
- Standard: PHENOL (utilized in hospitals)
- Vinegar → substitute for household bleach
 - QUATS o Observe signs of growth:
o Quaternary Ammonium Compounds ▪ turbidity, hemolysis, pellicle formation
o Ex. Benzalkonium chloride o Brucellosis: 3-4 weeks
o Neutralized by: o Leptospirosis: 8 weeks
▪ UK Acid, Sorbic Acid, Tween 80 + Lecithin, - Collect in 3 tubes; use TUBE #2 for microbiology
Lubrol W + Lecithin - Collected thru LUMBAR TAP
CSF
- Process ASAP; Store at 37degC; Transport at RT
- Use Sediments: for culture and smearing
PRIORIZATION OF SPECIMENS - SOC: Clean-Catch Midstream
1 Critical/Invasive CSF, Amniotic Fluid, Blood, - Unable to void: Catheterized
Pericardial Fluid, and Heart Valves
- Anaerobic culture: Suprapubic Urine
2 Unpreserved Feces, Sputum, Wound Drainage
- Usual Request: Culture and Sensitivity to rule out UTI
3 Quantitation Catheter tip, Urine, Tissues for quantitation o #1 cause of UTI: Community-Acquired E.coli
4 Preserved Urine, Feces, Swab in holding medium o UTI in young females: S. saprophyticus
5 Batch Processing Sputum, AFB Culture URINE o Other UTI-causing: Klebsiella, E. faecalis
- Perform COLONY COUNTING using Calibrated Loop
o Determines # of colonies/ mL of urine
SAMPLE PREPARATION
o Considered UTI >100,000 cfu/mL
- Detection of bacteremia and septicemia
o DF = if 1uL : 1000; if 10uL: 100
- Blood pathogens: [EPS]
- Formula:
o E. coli
o # of colonies counted x DF = colony count/mL of urine
o P. aeruginosa
- Bartlett’s Classification
o S. aureus
o >25 PMNs <10 SECS = Acceptable; sputum
- Biomarker for fungemia: C. albicans
o <25 PMSs >10 SECS = Reject; repeat collection; saliva
- Potential biomarker for sepsis: Procalcitonin and CRP
- For MTB, we do DECONTAMINATION AND DIGESTION
- Cleansed:
o Decontamination → remove Normal flora
o 70-95% Isopropanol + Iodine scrub + Alcohol rinse
o Digestion → to liquefy and remove trapped organisms
o Chlorhexidine maybe used instead of iodine
o Gold Standard: L-cysteine NAc + NaOH
- Common contaminants: [VeDa]
BLOOD ▪ L-cysteine Nac → digest
o C.acnes, S.epidermidis, Diptheroids, Viridans strep
▪ NaOH → digest and decontaminate (both if
- Dilution of blood to medium SPUTUM
used alone; but if incorporated=
o 1:10 (media: TSB, BHI, Brucella broth)
decontaminant only)
o AC: 0.025% Sodium Polyanetholsulfonate (SPS)
o Other agents:
o But, inhibits:
▪ Z-TSP
▪ Neisseria, Gardnerella vag., P.anaerobius,
• (Zephiran [dig], Trisodium phosphate
Streptobacillus monoliformis
[dec])
• Treat with 1% Gelatin
▪ 4% NaOH
- NOT used for culture: EDTA
▪ Cetylpyridium chloride sodium chloride
- AC for Viral culture, but inhibitory to G+ and yeast → HEPARIN method – inc. shelf life of sputum for 8 days
- Culture bottles held routinely for 7 days;
 ▪ 5% oxalic acid → added to specimen with GRAM STAINING
possible pseudomonas Purpose Reagent Gram (+) Gram (-)
o MTB: BSL-3; BSC-Class 2 Primary Stain/
Crystal Violet Violet Violet
Initial Stain
- ID of Streptococcus Pyogenes/ Group A = major throat Mordant Gram’s Iodine Violet Violet
pathogen Alcohol,
- Viridans strep. = Major throat flora Decolorizer Acetone, Violet Colorless
- NPS detect Alcohol-Acetone mixture
o carrier state of Neisseria meningitidis Counterstain/
Safranin Red Violet Red
▪ If the patient is symptomatic to N. meningitidis Secondary Stain
THROAT
use CSF Auramine-Rhodamine /
SWAB/ ACID FAST Ziehl Neelsen/Hot Kinyoun’s/Cold
o ID of B. pertussis and H. influenzae Fluorochrome
NPS STAINING (OIO) (OIO)
- For bacterial culture= use DACRON, CALCIUM ALGINATE (LPO)
o Toxic to Neisseria [CC] : Primary Carbolfuchsin Carbolfuchsin Auramine-Rhodamine/
▪ Cotton swab and Cold Temperature TRUANT Stain
• Treated with Charcoal (allowed) Mordant Heat/Steam Wetting Agent (-)
- For viral culture = use DACRON, COTTON, RAYON FIBERS Decolorizer 3% Acid-Alcohol 3% Acid-Alcohol 0.5% Acid-Alcohol
o Toxic to viruses: Calcium Alginate (inhibits replication) Counterstain MB/ MC MB/ MC 0.5%
- Must be processed within 2 hours Potassium permanganate
- Transport media: CARY BLAIR Result AFB (+) → Red against blue background AFB (+) → Yellow against
- Detection of GIT pathogens: ENTEROBACTERIACEAE AFB (-) → Blue/Green in blue background black background
o Routinely screened for [SaShiCam] Other methods of Acid-Fast Staining:
STOOL
▪ Salmonella, Shigella, Campylobacter - Pappenheim’s (urine); M. smegmatis = BLUE; M. tuberculosis = RED
- Rectal Swab → collected if stool is not possible - Baumgartens (tissue); M. leprae = RED; M. tuberculosis = BLUE
- # of quadrants = 4 [on MAC,EMB,HEA, SSA] - Fite Faraco’s; M. leprae → use of Hematoxylin as Counterstain
- Gram staining NOT usually done Additional Info:
SPECIAL STAINS - Ziehl-Neelsen → preferred for DSSM (Sputum)
Capsular Stains Hiss, Anthony’s Tyler, Muir - Kinyoun → preferred for Histology (Tissues)
Spore Stain Dorner’s, Schaeffer and Fulton, Wirtz and Conklin - Acid-Alcohol → HCl + Ethanol
Flagella Grays, Fisher and Conn, Leifson - Modified Kinyoun → for Cryptosporidium; Acid-Alcohol = Sulfuric Acid + Ethanol
Metachromatic Albert’s, Neisser, Ljubinsky, Ponder,
Granules Methylene blue, Lindergran, Burke’s Technique
Polar Bodies Wayson stain, Methylene Blue
Spirochetes Levaditi’s
 Consistency LIQUID SEMI-SOLID SOLID BIPHASIC KIRBY BAUER DISK DIFFUSION METHOD
Solidifying Media: MHA pH:7.2-7.4 Depth:3-5mm Antibiotic disk: 6mm
0% 0.5-1.% 2-3% -
agent/ Agar - subculture 4-5 colonies into TSB at 35-37°C for 3-5 hours
Liquefiable: - Mcfarland Standard: 99.5mL of 1% H2SO4 + 0.5mL of 1.175% BaCl
- EMB - Equivalent: 1.5 x 108 cfu/mL
TSB
- MSA HBT (G.vagina) - Compare turbidity; and if it’s aligned with the standard then we proceed in
APW
Examples SIM Castaneda streaking
BHI
Non-Liq.: (Brucella) - If too turbid: we can NSS or distilled water
Nutrient
- Rice - In streaking, we do OVERLAP STREAKING/LAWN TECHNIQUE
Medium - Wait for 3-5mins before application of antibiotic disk
Liquefiable → it can be melted after being solidified - If the MHA is 150mm = 12 disks; if 100mm = 5 disks
Non-liquefiable → it cannot be melted after being solidified - Distance of disks from the center = 24 mm
Dissolve with dH20 or deionized H20 - Distance between 2 disks = 15 mm
TUBE: Weigh – Dissolve – Dispense – Sterilize [TS] - Invert plates and incubate in 35-37°C for 16-18hrs; in case MRSA: 35°C, Aerobic
PLATED : Weigh – Dissolve – Sterilize – Dispense [PD] - Measure Zone of Inhibition through the use of Ruler or Caliper
PURPOSE - If Media is with blood= measure on top, and uncovered
General Contain only what is needed - Streptrococci = MHA + 5% Sheep Blood’s Agar
Enhance bacterial growth/yield - Too light inoculum; thin
Enrichment
Ex. APW, Selenite Broth, Tetrathionate Broth - Dry
Fastidious; contains blood False (S)
Enriched - Acid pH → affects Tetracycline, Methicillin, Novobiocin
Ex. CAP, BAP - Alkaline pH → affect aminoglycosides/erythromycin
Promote growth of particular organism - Too heavy inoculum; thick
Prevent growth of others False (R) - Moist
Selective
Have inhibitors (ex. Antibiotics) - Prolonged incubation
Ex. MAC (Crystal Violet), CTBA (Potassium Tellurite) - Mixed culture → source of error
Differentiate organisms that are growing together - Improper storage of antibiotic disks
Differential
Ex. MAC (LF or NLF) , TCBS (Sucrose or Non-sucrose Fermenter) o If opened → 2-8°C [Ref]
Selective and MAC, EMB, TCBS o If unopened → -20°C in non-frost freezer
Differential - Swarming → ignore
Maintain moist environment - If Sulfonamide is utilized, and 2 concentric zones are seen → Measure outer zone
Transport Keep organism alive during transport
ANTIMICROBIAL AGENTS
Ex. JEMBEC and Transgrow (Neisseria), Cary Blair (Stool), Amies
Beta Lactams = Penicillin, Carbapenems, Cephalosporins
Biochem Test Aids in bacterial ID Cell Wall Inhibitors
Glycopeptides = Vancomycin (DOC for MRSA)
Media Ex. TSI, LIA
Macrolides = erythromycin
Susceptibility MHA
Protein Synthesis Aminoglycosides = gentamicin, tobramycin
Media
Inhibitors Tetracycline = doxycycline
SIM
Chloramphenicol (Aplastic Anemia)
Motility
 Beta-Lactamase Tazobactam, Sulbactam, Clavulanic acid (GasPak Jar) Uses Palladium Catalyst → maintain anaerobiosis
Inhibitors Most common failure → inactivation of catalyst
Sulfonamides Sulfamethoxazole (SXT) Indicators:
Nitrofurantoin Treatment for UTI; inhibits bacterial enzyme - Methylene Blue → BLUE
Inhibition of DNA activity; - Resazurin → PINK
Quinolones
Levofloxacin, Ciprofloxacin - (+) Color formation
EQUIPMENT MONITORING - (-) Colorless
Incubator, water bath, refrigerator, Temperature Check Daily Microaerophilic/ 5% O2, 10% CO2, 85% N2
Freezer, Heating block Capnophilic For Neisseria, Campylobacter
Autoclave – efficiency/ spore test Weekly (Candle Jar)
Autoclave Temperature Daily/ Every Load OTHER INFO:
GASPAK Jar Every load/ Every Use Benchmarking Peer comparison
Centrifuge Every 6 months Brightfield Light microscopy
Microscope 4x a year/ as needed microscope
Weighing balance Weekly Purpose of - Allow growth of facultative anaerobes
Oxidase, Catalase, Gram Stain Weekly boiling - Growth at bottom : Strict Anaerobes
REPORT IMMEDIATELY : Critical Values thioglycolate - Growth at top : Strict Aerobes
- (+) POSITIVE: - Dispersed/Diffused Growth: Facultative Anaerobes
o Blood Culture, CSF gram stain or culture, cryptococcal antigen test or Handwashing Most effective way of to stop chain of infection
culture, blood smear of malaria, Streptococcus pyogenes in sterile site, Universal ALL HUMAN BLOOD AND BODY FLUIDS
Positive Acid-fast smears/ Mycobacterium culture, Strep. Agalactiae or Precaution
herpes simplex virus from genital site of a pregnant woman at term, Standard POTENTIALLY INFECTIOUS
Detection of significant pathogen (Bordetella pertussis, Brucella, Precaution
Legionella) Donning Gown-Mask-Googles/Faceshield-Gloves
PCR Doffing Gloves-Goggles/Faceshield-Gown-Mask
Denaturation 95°C HOT/COLD AFB GRADING
Annealing 50-56°C 0 No AFB in 300 fields
Elongation 72°C +n 1-9 AFB / 100 fields
BACTERIAL IDENTIFICATION +1 10-99 AFB/ 100 fields
Manual Method Prone to contamination; not performed +2 1-10 per field (min. 50 fields)
Semi-automated Analytical Profile Index-API +3 >10 per field (min. 20 fields)
API 20E, API 20A FLUOCHROME AFB GRADING
Plastic strips and microtubes with biochemical substrates Negative 0
Biochemical substrates inoculated with pure culture Doubtful; repeat new specimen 1-2 AFB/ 70 fields
Automated VITEK 1+ 1-2 AFB/ 100 fields
MALDI-TOF 2+ 2-18 AFB/ 50 fields
CULTURE ENVIRONMENT 3+ 4-36 AFB per field [x2 ang 2+]
Anaerobic 5% CO2, 10% H2, 85% N2 4+ >36 AFB per field
Culture For anaerobes
 TESTS Purpose REAGENT RESULT
Staphylococcus and Micrococcus 3% Hydrogen Peroxide (+) Vigorous Bubbling/Effervescence
Catalase Test
VS Streptococcus
S. aureus VS Rabbit’s Plasma (+) Clot formation
Coagulase Test S. epidermidis and saprophyticus (-) No clot after 20 hours incubation [4 hours at
37°C; 16 hours at RT]
S. aureus MSA (+) Yellow halo around colonies
Mannitol Fermentation 7.5% Salt
Phenol Red
S. aureus HCl Precip. (+) Clearing of agar
DNAse Test
Thermonuclease Test Dye Method Methyl green: (+) Clear Zone
Toluidine Blue: (+) Pink Zone
Microdase: Disk 6% TMPPD in DMSO (+) Blue Color
Oxidase Test Modified Oxidase: Filter paper
Staphylococcus VS Micrococcus
Neisseria 1% TMPPD without DMSO (+) Purple Color

Test for beta lactamase production
Cephalosporinase test
CHROM AGAR
Detection of
MRSA/ORSA
CEFOXITIN DISK DIFFUSION TEST
Induces expression of PBP2A
OXACILLIN SCREEN AGAR
PCR
Gold Standard for MRSA Detection
Leucine Amino Peptidase Detect G(+), Catalase (-) → usually
(LAP Test) Streptococci
Capsular Swelling Test/ Detect capsule
Neufeld Quellung Test
S. pneumoniae VS Viridans
Bile Solubility Test
S. agalactiae VS S. pyogenes
PYR Test
Enterococci VS Non-Enterococci
Cefinase Disk
Substrate: Nitrocefin
MHA + 6ug Oxacillin with 4% NaCl
Substrate: Leucine-B-naphthylamide
Reagent: Cinnamaldehyde
In Media: Sodium Desoxycholate
In Broth: 2% Sodium Desoxycholate
Substrate: Pyrrolidonyl-Alpha-naphthylamide
Reagent: Para-dimethylaminocinnamaldehyde
(+) Pink to Red Color
MRSA: Rose Mauve colony, growth (resistance)
Non-MRSA: No growth (susceptible),
Colorless/blue colonies
MRSA (+) Growth Inhibition
(+) Red Color
(-) No color/ Slight Yellow
(+) Organisms with Capsule
(+) Lysis of colonies
(-) Intact Colonies
(+) Clearing/ Partial Clearing
(-) Turbidity
(+) Red Color Development
(-) Orange or no color Change

Hippurate Hydrolysis Test
CAMP TEST
REVERSE CAMP TEST
DICK’S TEST
SCHULTZ-CHARLTON
BLANCHE PHENOMENON
Bile Esculin Test
Salt Tolerance Test
CHO Utilization Test
Butyrate Disk Test/ Tributyrin Test
Heat or
Ethanol Shock Test Treatment
Ascoli Test
Anton’s Test
Modified ELEK Test
Animal Inoculation/
Guinea Pig Lethal Test
SHICK’s Test
S. agalactiae VS S. pyogenes
Hydrolysis of Sodium Hippurate by
Hippuricase/ Hippurate hydrolase
S. agalactiae
L. monocytogenes
C. perfringens
Susceptibility test for Scarlet Fever
Determine if RASHES are due to
Scarlet Fever
Screening test for Enterococci and
Group D-Non Enterococci/ S.bovis
group
Enterococci VS Group D-Non
Enterococci/ S.bovis group
Detect capacity of organism to
utilize carbohydrate
Rapid ID of Moraxiella catarrhalis
Stimulate spore formation
Serologic Test for B. anthracis
Virulence test for Listeria
monocytogenes
Toxigenicity test for C. diphtheriae
Toxigenicity test for C. diphtheriae
Immunity Test; for Patient at risk of
developing diphtheria
To detect BENZOIC ACID: Ferric Chloride (+) Purple Color
To detect GLYCINE: Ninhydrin Reagent
Known organism: S. aureaus (+) Enhanced hemolysis; arrow head
(-) no enhanced hemolysis
Known organism: Rhodococcus equi (+) Block-type hemolysis
Known organism: S. agalactiae (+) Enhanced hemolysis; arrow head
(-) no enhanced hemolysis
(+) Redness at the site of injection
Anti-erythrogenic Toxin (+) Fading/gradual disappearance of rash
Media: Bile Esculin (+) Blackening of Media
Indicator: Ferric Ammonium Citrate
6.5% NaCl (+) Growth [Enterococci]
Media: CTA (Cystine Trypticase Agas) (+) Yellow
Indicator: Phenol Red (-) Red
Subtrate: Bromo-chloro-indolyl-butyrate (+) Blue
70 degC for 30 mins/
80 degC for 10 mins
62-65 degC for 10-15 mins → B. anthracis
Culture is inoculated on conjunctival sac of rabbit (+) Purulent conjunctivitis
Filter paper strip with diphtheria anti-toxin + control at right (+) Line of precipitin
angle (+) Line of precipitin with Arc of identity
Unknown culture suspected of C. diphtheriae is streaked parallel [MAHON}
to + and – control
Incubate at 35 degC for 24-48 hours
0.3mL of isolated strain is injected subcutaneously to thigh of (+) Unprotected guinea pig dies within 2-3 days
two guinea pigs; one protected with 500 units of diphtheria with evidence of hemorrhage in the adrenal
antitoxin 18-24 hours before the st glands

Vero Cell Cytotoxicity Assay
Niacin Test
Nitrate Reduction Test
Iron Uptake Test
Arylsulfatase Test
Pyrazinamidase Test
Heat Stable Catalase Test
TWEEN 80 Hydrolysis Test
Tellurite reduction test
Salt Tolerance Test
T2H/TCH Susceptibility
Thiopene-2-carboxylic hydrazide
Growth in MAC without CV
Mantoux Test
Lepromin Test
Gold standard for Diphtheria toxin
production
MTB LJ Media (+) Yellow
Strip impregnated with Cyanogen Bromide
MTB Sodium Nitrate Broth
After incubation, we add:
- N-n-dimethyl-l-naphthylamide and sulfalinic acid (+) Initial RED
(-) No color change
Confirm (-) add:
- Zinc dust/ zinc powder (+) No color Change
(-) Red
Mycobacterium LJ Media (+) Rusty brown color
20% Ferric ammonium citrate
Detection of rapid growers Phenolphthalein media (+) Pink color
(+) M. fortuitum-chelonae 2N Sodium carbonate
Pyrazinamidase agar (+) Red/Pink color Band
1% ferrous ammonium SULFATE
To detect Heat Labile MTB Tween 80 Media (+) Bubbling
Heating of colonies at 68 degC, then
Add of 30% hydrogen peroxide
ID of M. kansasii = growth in 3 days Neutral Red (+) Pink color
MTB = 10-20 days
ID of M. avium (+) Black metallic tellurium
For ID of rapid growers & M. triviale 5% NaCl
To differentiate MTB from MBovis M. bovis → Susceptible
M. TB → Resistant
To ID M. fortuitum-chelonae
complex
Purified Protein Derivative (PPD) PPD injected intradermally Induration Size:
- Part of MTB (cell wall) (+) >10 mm = for Immunocompetent
(+) >5 mm = for immunocompromised
Von Pirquet/ Scratch Test
- Old Tuberculin -> Ointment (Old tuberculin + Lanolin) is rubbed against Skin (+) Redness
attenuated/killed MTB (-) Wheal disappearance within 30 mins
Skin test for M. leprae Observation of LEPRA CELLS (AFB within a macrophage) (+) Lepra Cells
Wade Fite Faraco Detect AFB in paraffinized tissues Uses Hematoxylin as counterstain

To detect if Haemophilus requires X Porphyrin → UV Light (+) Red fluorescence

factor for growth
D-ALA Test/ Porphobilinogen → KOVAC’s (PDAB) Reagent (+) Red color
Porphyrin Test To detect ability of organism to
convert D-ALA to Porphyrin and POSITIVE RESULT means Haemophilus DOES NOT
Porphobilinogen require X factor
 SPECIES OTHER NAME
Bacillus cereaus Fried rice bacillus
Bacillus subtilis Hay bacillus
Clostridium perfringens Gas Gangrene bacillus
C. welchii Frankel’s bacillus

Clostridium tetani Lollipop Bacillus

Drumstick bacillus
Tack head bacillus
Tennis racket bacillus
Clostridium botulinum Canned good bacillus
Von Ermengen’s bacillus
Corynebacterium diphtheriae Kleb Loeffler’s bacillus
Corynebacterium pseudodiphthericum Hoffman’s bacillus
Corynebacterium jeikeium JK Bacillus
Mycobacterium tuberculosis Koch’s bacillus
Tubercle bacillus
Mycobacterium leprae Hansen’s bacillus
Mycobacterium kansasii Cross-barred bacillus
Mycobacterium gordonae Tap water bacillus
Mycobacterium intracellulare Battey bacillus
Mycobacterium terrae Radish bacillus
Mycobacterium triviale V bacillus
Mycobacterium gastri J bacillus
Shigella dysenteriae Shiga bacillus
Shigella flexneri Strong’s bacillus
Shigella boydii New Castle Manchester bacillus
Shigella sonnei Duval’s bacillus
Klebsiella pneumoniae Friedlander’s bacillus
Escherichia coli Colon’s Bacillus
Burkholderia mallei Glander’s bacillus
Burkholderia pseudomallei Whitmore’s bacillus
Haemophilus influenzae Pfeiffer’s bacillus
Brucella abortus Bang’s bacillus
Bordetella pertussis Bordet Gengou bacillus
MYCOLOGY SPORES
Characteristics: SEXUAL SPORES Meiosis, With Nuclear Fusion
- Eukaryotic; No chlorophyll, obligate aerobes, saprophytic Ascospores Within sac-like structure (Ascus); usually fixed #8
- Inhibits water, solid and decaying organic debris, plant kingdom without roots and Basidiospores Within club-shape structure (basidium(
stems → referred as THALLOPHYTES Zygospores Undifferentiated; Identical cells
- pH 5.0 (acidic); Need moisture for growth; slow growers Oospores Differentiated; Non-identical cells
- Chitin (Cell Wall); Ergosterol (Cell Membrane) ASEXUAL SPORES Mitosis, Without Nuclear Fusion
- Monomorphic → exist as yeast alone or mold alone Conidia
- Dimorphic → exist as either yeast or mold; temperature sensitive Produced asexual spores singly or grouped by specialized
YEAST MOLDS vegetative hyphal strand called Conidiophore
- Unicellular; Fan - Multicellular; aerobic Microconidia Unicellular
- Round or Non-filamentous - Filamentous → produce hyphae Macroconidia Multicellular
- Asexually → budding - Colonies: Fluffy, cottony, wooly, and Arthroconidia/ Fragmentation of hyphal cells
- Colonies: moist, creamy, opaque, powdery Arthrospores Ex. Trichosporon, Geotrichum, Coccidioides
and pasty - Grows at RT (25°C) Blastoconidia/ Budding of hyphal cells
- Grows at 37°C - Microscopy for identification Blastosppores Ex. Candida
- Requires biochemical test for (demonstrate spores and hyphae) Enlargement of hyphal cells
differentiation Ex. Candida
Aseptate (Coenocytic) No cross walls Chlamydoconidia/ Types:
Septate – hyphae divided by filaments All are septate except Chlamydospores - Intercalary = within
Cross walls
ZYGOMYCETES (Rhizopus, - Sessile = at side/ lateral
Absidia, Mucor) - Terminal = at end
Hyaline/ Moniliaceous Aspergillus Sporangiospores
Fusarium Produced thru sporangium
Rhizopus Ex. Zygomycetes (Rhizopus, Absidia, Mucor)
Pigmentations Dematiaceous / Phaeoid Alternaria SPECIMENS
Bipolaris Respiratory Tract - Most common specimen collected for fungal culture
Cladosporium Secretions - Recovery of agents causing systemic mycoses (Histoplasma
Phialopora capsulatum)
Spiral (coiled hyphae) T. mentagrophytes - Ex. Sputum, Bronchial washings
Nodular shapes M. canis CSF - For ID of C. neoformans in fungal meningitis
Racquet – with club shaped areas Epidermophyton floccosum - Collected sample is processed thru filtration or
Shapes Pectinate body M. audinii sedimentation
Favic chandelier/ Antler-curved, freely T. schoenlenii - Use 0.45 um filter attached to sterile syringe
branching T. violaceum BLOOD - BACTEC → rule out disseminated infections
Rhizoids – rootlike Rhizopus - Lysis Centrifugation system → recovery of dimorphic fungi
Vaginal specimens - Transported within 24 hours of collection
and Urine - Vaginal secretions
o screen for yeast; incubated at 30degC for 7 days
Hair, Skin, Nails -
collected for recovery of TINEA - Background: GREEN
-
Infected Hairs - For Tissue/ histologic Specimen
o Plucked using forceps Acid Fast (Kinyoun’s) - Demonstrate Nocardia and B. dermatitidis
o Fluoresce under wood’s lamp - Better visualization of Blastomyces
o Sterilized paper envelopes Giemsa or Wright - Demonstrate YEAST of H. capsulatum
- Surface of the skin Stain
o Disinfected with spirit (methylated alcohol) Gomori - Detect fungal elements in histologic section
o Scrape the advancing edge of lesion thru blunt Methenamine Silver - General color: Brown to black
forceps and in sterilized paper envelopes o Outlines of fungi → Black
- Discolored or Hyperkeratotic areas of nails o Integral parts → Pink, Black
o Scraped o Background → Light Green
o Diseased nail clipping may be collected in sterilized - Candida and Aspergillus are missed out by H&E; therefore,
paper envelopes utilization of GMS is essential for tissue histology
Tissue, BM, and - Must be MINCED or use High-speed Blender Acridine Orange - For TINEA VERSICOLOR (by M. furfur)
Sterile Body fluids - Grown on agar; not on broth o Fungal element → green fluorescent
Eye - Corneal scrapings o Epithelial cells → Orange
- Eye discharge Fontana Masson - For determine if Moniliaceous/Hyaline or
MICROSCOPIC EXAMINATION H&E Dematiaceous/Phaeoid
TEMPORARY MOUNT Mayer’s - For C. neoformans
10-20% KOH - Detect fungal elements of SKIN, HAIR, NAILS Mucicarmine
- Skin and Hair = 10% Gridley’s Stain - Yeasts and hyphae → dark blue-pink
- Nails = 20% (hard tissues) - Tissues → deep blue
- Purpose of heating: increase rate of clearing - Background → Yellow
- Alternative: Sodium hydroxide Wood’s Lamp - Detect fluorescence
Lactophenol - Stain and Preservative - UV Light
Cotton Blue o Lactic Acid → Preservative CULTURE MEDIUM
o Phenol → Killing Agent Saboraud’s Dextrose - General purpose media
o Cotton Blue → Staining Agar (SDA) - pH: 5.6 (Fungi are acidophilic)
India Ink/ - Demonstrate CAPSULE of C. neoformans Dermatophyte Test - Screening media for Dermatophytes
Nigrosin Medium (DTM) - Indicator: Phenol Red
Calcofluor - Fluorescent dye; requires fluorescent microscope Mycosel/ - Screening media for Dermatophytes
White Stain - Demonstrate CHITIN in cell walls Mycobiotic Agar o SDA + Chloramphenicol → Bacterial
- Best for direct microscopy o SDA + Cycloheximide → Fungal
PERMANENT MOUNT Cornmeal Agar - CONFIRMATORY: Demonstrate CHLAMYDOSPORE
Gram Stain - Uses CV + Ammonium Oxalate formation of Candida albicans
(Hucker’s Mod.) - All fungi are Gram (+) - Cornmeal agar + 1% Glucose: Differentiate T.
- For tissue/ histologic specimen mentagrophytes (-) and T.rubrum (+) = Red pigment
Periodic Acid Schiff - Detect details of fungal elements Cornmeal Agar with - Presumptive ID of Candida albicans
- (+) pink-red/ Bright magenta
 Tween 80 and Temperature Slides - Differentiate Candida albicans from Candida dublinensis
Trypan Blue o (+) growth at 42 degC → Candida Albicans
Czapek Agar - Aspergillus o (-) growth at 42 degC → Candida dublinensis
Niger Seed/ Bird - For C. neoformans Carbohydrate - For ID of yeast isolate; free from carbohydrates
Seed/ Staib’s Assimilation Test - (+) Growth of Yeast around the disk
Medium o Indicates that yeast is able to utilize carbohydrate
Potato Dextrose - Demonstrate pigment production of T. rubrum as sole source of carbon
Agar - Red pigment SUPERFICIAL MYCOSES (Skin; No Immune Response)
Potato Flake Agar - Recovery of saprobic and pathogenic fungi - Malassezia furfur
Cottonseed Agar - For Blastomyces o Needs olive oil for culture
Pityriasis Versicolor/
SABHI (Saboraud’s - For Blastomyces and Histoplasma o In culture: Bowling Pin appearance
Tinea Versicolor
Brain Heart Infusion - Yeast conversion o In microscopy: Spaghetti with Meatballs
Agar) o Skin discoloration (body)
Brain Heart Infusion - For yeast - Exophiala werneckii/ Hortaea werneckii
Tinea Nigra/
Agar o Dark brown to black painless patches at soles of
Keratomycoses
Rice Medium - Differentiate M. audinii (-) from M. canis (+) feet and palms of hands
Nigricans Palmaris
Urea Agar - Differentiate T. mentagrophytes (+) from T. rubrum (-) o In culture: Moist-shiny black, yeast-like colonies
- ID of Cryptococcus, Trichosporon, Rhodotorula spp. - Mostly in tropical areas
Piedra hortae
Casein Medium - Nocardia - Affects SCALP HAIR
(Black Piedra)
Malt Extract Agar - For M. furfur PIEDRA/ - Black color, Hard ascospores (sexual)
Hay Infusion Agar - NOT a fungal medium Tinea Nodosa/ - Mostly in temperate areas
SPECIAL TESTS Beigel’s Dse/ Trichosporon - Affects FACIAL, GENITAL, AXILLARY
Hair Perforation/ Chignon Dse beigelii HAIRS
- Differentiate T. mentagrophytes (+) from T. rubrum (-)
Baiting Test - Specimen: Hair (White Piedra) - Creamy-colored, Soft Arthrospores
- (+) Result: (asexual)
Rapid Urease Test - Hair perforation; Conical shaped/ V perforation CUTANEOUS MYCOSES (Skin, Hair, Nails = Keratinized Tissues)
- Pink-Red coloration SKIN HAIR NAILS
L-DOPA Ferric - Rapid ID of Cryptococcus Neoformans Tinea corporis Tinea Capitis Tinea unguium
Citrate Test - (+) Black Color - Bodily skin - Scalp hair - Onychomycosis
Germ Tube Test - Presumptive ID of Candida Albicans - BUNI Tinea barbae - Nail and nail bed
- SBA Colonies + Serum (any source) → incubate 30 mins → Tinea cruris - Facial hair
place to slide and microscopy - Inguinal area Tinea favosa
- (+) hyphae-like extensions of young yeast cells - JOCK-ITCH/ - Hair follicles
Exoantigen Test - Confirm systemic mycoses HADHAD
- (+) Presence of Precipitin Band Tinea pedis
- Coccidioides immitis → CF, TP, HL Antigens - In between of Foot
- Histoplasma capsulatum → H or M band (Both) - ATHLETE’S FOOT
- Blastomyces dermatitidis → A band Tinea manuum
 - In between of - Favic “antler” hyphae
Fingers Trichophyton violaceum - Violet purple colonies
MICROSPORUM microSOFT → SKIN and HAIR) - Favus/ port wine-colored colonies
TRICHOPHYTON TRI (3) → SKIN, HAIR, and NAILS SUBCUTANEOUS MYCOSES (Deep Layer, Dematiaceous; soil; trauma)
EPIDERMOPHYTON Pid (pedicure) + Derm (skin) → SKIN and NAILS Sporotrichosis - Rose gardener’s Dse; Occupational Hazard
Affects the SKIN AND NAILS Sporothrix schenkii - Yeast: Cigar-shaped yeast
Trichophyton - Mold: Flowerette-arrangement
Trichophyton rubrum Epidermophyton flocossum Phaeohyphomycosis - Infects Skin, Brain, and Lung of Immunocompromised
mentagrophytes
- tear shaped or - No microconidia - Globose Px
clavate - Macroconidia microconidia and - Example: [ABCDE]
microconidia resembles Beaver’s arranged in Grape- o Alternaria, Bipolaris, Culvularia, Dreschlera,
- Birds-on-a-fence Tail like clusters Exophiala
appearance - (+) Hair baiting Chromoblastomycosis/ - Cauli-flower like lesions
- (-) Hair baiting - (+) Urease Chromomycosis/ - Diagnose thru presence of:
- (-) Urease - (-) Pigment Mossy Foot Dse/ o Sclerotic bodies/ copper pennies/ fission body/
- (-)Pigment Hematomycosis Muriform cells
Affects the HAIR - Examples:
o Phialophora verrucusom
Microsporum canis - Thick walled multiseptated o Cladosporium carrionii
(zoophilic) - Large-spindle shape macroconidia o Fonsecea pedrosoii
A→H - Yellow green fluorescence Rhinosporidosis - Lesions in nose and pharynx
- RICE medium (+) Rhinosporidium siberi
Microsporum audouinii - Bizarre shaped macroconidia Keloidal Blastomycosis/ - Dimorphic fungi
(anthrophilic) - Apple green fluorescence Lobomycosis
H→H - RICE medium (-) Lacazia loboi
- Favus; Epidemic Ringworm Mycetoma/ - Granulomatous tumor of subcutaneous tissues
- Culture media: SDA + PDA Madura Foot - Tumor-like deformities
Microsporum gypseum - (-) fluorescence
(geophilic) Bacterial Actinomyces, Nocardia,
Trichophyton - Favus, Embedded stubs (SANA) Streptomyces, Actinomadura
verrucusom - Microconidia: Balloon shape White Grain Pseudoallesheria boydii, Acremonium
- Macroconidia: Rat Tail/ String Bean Black Grain Exophiala jeamselmi, Madurella mycetomatis
Trichophyton tonsurans - 2nd most common cause of Epidemic Tinea Capitis SYSTEMIC MYCOSES (Inhalation; lungs; Molds [ Infx ]; Yeast [ Dx ]
- Requires THIAMINE
Blastomycosis - Blastomyces dermatitidis
- Microconidia: Balloon-shaped; Matchstick form
North American o VF: BAD-1 (adhesion, immune regulatory)
- Black-dot Ringworm Chicago o Mold: round or pyriform conidia; Lollipop
Trichophyton - Favus alopecia Gilchrist’s o Yeast: large with single bud
schoenlenii - Mousy odor scalp Missouri River Valley
 -
Coccidioides immitis o Oral thrush → oral infection of
o Most virulent of all mycotic agent immunocompromised hosts; creamy patches
o Major biohazard in laboratory o Vulvovaginitis, thrush, Moniliasis
o VF: extracellular proteinase - Forms germ tube, Blastoconidia and chlamydospores
San Joaquin Valley
o Cob-web like colonies o Germ tube = (+) C. albicans; (-) C. tropicalis
o Mold: Barrel-shaped arthroconidia with o Corn meal Agar = confirm C. albicans
alternating disjunctor cells o 42 degC = (+) C. albicans; (-) dublinensis
o Yeast: thick-walled spherule with endospores - Other types:
- Histoplasma capsulatum o C. tropicalis → 2nd most common
o Survive within macrophage o C. glabrata → fungemia
Histoplasmosis
o MOT: Inhalation of spores from bat droppings o C. parapsilosis → nosocomial infections
Darling’s
▪ Found in bats and birds feces Aspergillus - Aspergillus niger
Cave
o Yeast: Yeast within mononuclear cells o Black pepper effect; fungus ball
Spelunker’s
o Molds: Knobby Tubercle Macroconidia Pulmonary Dse - Aspergillus fumigatus
Reticuloendothelial-
o VF: Otomycosis o Most common cause of pulmonary aspergillosis
Cytomycosis
▪ Intracellular growth Onychomycosis o Found in compost piles and soil of potted plants
Ohio Valley
▪ Thermotolerant Fungus Ball o Green color in media
▪ Calcium-binding protein Aspergilloma - Aspergillus flavus → yellow
o We stain the cell with yeast Farmer’s Lung - Aspergillus terrae → brown
- Paracoccidioides brasiliensis Talaromyces marneffei - Animal reservoir: Bamboo Rat
o VF: Estrogen Binding Protein Penicillium marnefei - Yeast: sausage shaped
South America o Yeast: Ship’s wheel, Mariner’s Wheel, - Mold: Flask-shaped phialides, paint brush-like
Luts Splendore Almeide Navigator, Pilot, Parent cell surrounded by Talaromycosis - TB like symptoms in HIV patients in SEA
daughter cells, Mickey Mouse cap - Atypical fungus
o Mold: Terminal chlamydoconidia - Formerly: Pneumocystis pneumonia
OPPORTUNISTIC INFECTION (Immunocompromised) Pneumocystis jirovecci - Pneumonia in AIDS from immunocompetent animals
- Major cause of opportunistic infection in Px with AIDS Pneumocystis carinii - Sample: BAL
Cryptococcus
- MOT: inhalation of spores in bird droppings - Gomori methenamine silver → best for cyst; not for
neoformans
- Found in bird feces/pigeon droppings and soil trophozoite
o (+) Capsule in India Ink Zygomycetes - Organisms: Rhizopus, Absidia, Mucor
Torulosis
o (+) Phenol Oxidase → melanin production - Fast growers; Lid lifters
Torula Meningitis
o (+) Urease → 2-3 days; uses Benzalkonium Rhino sinus - Aseptate Hyphae
European Blastomycosis
chloride (to release urease) Sinus orbital/ - (+) growth on SDA without cycloheximide
Bussbusche Dse
o (-) Nitrate Craniofacial
- Most common cause of fungal diseases worldwide Zygomycosis
- MOT: Direct invasion or dissemination Other opportunistic fungi:
Candida albicans
- Normal flora; major cause of fungemia - Fusarium spp. (hyaline) → fungi imperfecti; normal mycoflora in commodities RICE
- Disease to any site; Disease: - Geotrichum → wound infection and oral thrush; hockey stick-shaped arthroconidia
on corn meal agar
 - Penicillium (hyaline) → keratitis, external ear infection SARS COV2 ACE 2
- Aspergillus → A. fumigatus (most common to produce AFLATOXIN)
- Zygomycosis → Mucor (common lab contaminant) 2. Penetration
- Acremonium → mycetoma, cornmeal and nail infections o Engulfment of virus thru endocytosis
VIROLOGY o Uncoating or release of genetic material
- Obligate intracellular organism; Acellular; DNA or RNA 3. Biosynthesis
- Haploid, except Retrovirus (diploid) o Synthesis of viral proteins/ Eclipse Phase/ Replication
- Lack genes and enzymes necessary for energy production o Entry to either Nucleus (DNA) or Cytoplasm (RNA)
- Resistant to antibiotics; 200-300nm in diameter; o Some components are made in tremendous excess
- virulent or temperate 4. Morphogenesis
VIRAL STRUCTURE: Virion (entire viral particle) o Formation of VIRION from all viral structure
Genome/ Center; contains genetic material (RNA or DNA) 5. Release to extracellular fluid
Nucleic Acid Core SAMPLE SPECIMEN
Capsid Surrounds or encloses the genome Throat Swab – Influenza virus
- Cube-like → Icosahedral Respiratory Syndrome Nasal Washings – Parainfluenza virus
- Rod-like → Helical Nasopharyngeal Aspirate -- RSV
- Complex → Pox Rectal Swab – Norwalk Virus
Gastroenteritis
Nucleocapsid Genome + Capsid Stool – Rotavirus
Capsomeres Morphological units of capsid; made from protomers Skin scrapings/ lesion swab – VZV
Skin Lesion/Rash
Vesicular fluid – HSV
Protomers Smallest functional unit to build capsid; make capsomeres
Congenital Infection Urine – CMV
Outer Membrane Enveloped (composed of lipids and polysaccharides)
Encephalitis/ Blood and CSF – Togavirus
- Sensitive to harsh/unfavorable environment
Aseptic Meningitis
Naked/Non-enveloped
- Either resistant/stable in adverse environment DIAGNOSIS
Protein spikes For transfer of genetic material Direct Microscopic Exam/ Cytology and Histology
1. Light Microscopy → for POX Virus
STEPS IN VIRAL REPLICATION
2. Phase Contrast Microscope → for Viral Inclusions
1. Attachment/ Adsorption
a. Viral Inclusions – virus particles; seen in either nucleus (DNA) or cytoplasm
o Interaction between viral and host cell receptor
(RNA) of infected cells
o Virus exhibits tropism (react with specific receptor only)
i. Rabies – Negri Bodies
HIV CD 4
ii. Yellow fever virus – Torres Councilman
EBV CD 21
iii. Fowl Pox virus – Bollinger bodies
MERS COV CD 26
iv. Variola and Vaccinia – Guarneri Paschen bodies
Measles CD 46
v. Molluscum contagiosum – Henderson Patterson/ Lipschultz
Rabies CD 56 - Acetylcholine Receptor b. Koilocytes in Pap stained smear → Diagnostic for HPV
Poliovirus CD 155 (Pcr) i. Koilocytes – atypical squamous and atypical nucleus surrounded
Rhinovirus ICAM-1 with Non-staining Halo (Perinuclear halo) = Abnormality
Vaccinia Epidermal growth factor c. Tzank Smear → detects VZV and HSV Inclusions
Influenza Sialic Acid i. VZV – Cowdry Type A bodies
 ii. Routine Stain: GIEMSA + PAPs QUANTIFICATION OF CPE
iii. For EM : Potassium phosphotungstate or Uranyl Acetate Negative Uninfected monolayer
3. Electron Microscopy → for Viral morphology Equivocal (+/-) Atypical alteration
Viral Culture/ Cell Culture (GOLD STANDARD; shell vial; laborious) 1+ 1% - 15% cells exhibit CPE
I. Primary Cell Culture 2+ 25 – 50 %
a. Source: Normal Mature Adult Cells 3+ 50 – 75 %
b. Passage: Once or Twice 4+ 75 – 100 %
c. Most common: KIDNEY CELLS (rhesus monkey or rabbit kidney) VIRAL ANTIGEN DETECTION
i. Primary Monkey Kidney Cells → excellent recovery of MYXO, - Direct and Indirect Immunofluorescence
ENTERO, may support growth of ADENO, RSV, MEASLES o Use of fluorescein labelled antibody
II. Diploid / Semi-Continuous Cell Line o for RSV, PIV, Adeno, Influenza, HSV, CMV
a. Source: Embryonic or Fetal Cells (Actively Dividing) o FITC Dye (Fluorescein Isothiocyanate)
b. Passage: 50x - EIA, Latex Agglutination
c. From: Embryonic tissues (fibroblasts of lung or foreskin) - ELVIS → Enzyme-linked Virus Inducible System for HSV
i. Ex. Human Fetal Diploid → isolation of VZV and HSV FITC FLUORESCENCE INTERPRETATION
ii. Other example: MRC5, WI-38 Negative No Apple Green
III. Heteroploid/ Continuous Cell Line 1+ Faint Apple Green
a. Source: Malignant Tumor/ Cancer/ Immortalized Cells 2+ Apple Green
b. Passage: Indefinitely
3+ Bright Apple Green
i. Examples: Hep-2 continuous cell line → for adenovirus, HSV and
4+ Brilliant Apple Green
RSV
DNA VIRUSES
ii. HeLa, Cervical cancer cells Vero, Hep2 Laryngeal Cancer Cells, LLC-
All are dsDNA, except Parvovirus (smallest)
MK2, MDCK, A549 Lung Carcinoma cells; KB
All are Icosahedral, except Pox virus
CYTOPATHIC EFFECT
All are enveloped, except PPAP Papilloma-Polyoma-Adeno-Parvo
- Change in the nucleus/ cytoplasm of an infected cell due to presence of virus
All replicates in nucleus, except Pox virus (largest)
- Examined thru INVERTED LIGHT MICROSCOPE
RNA VIRUSES
- Examples of CPE:
All are ssRNA, except Reoviridae
o Pyknosis → condensation of nucleus (smaller) ex. PICORNA
All are Icosahedral, except BROPaFC Bunya-Rhabdo-Ortho-Paramyxo-Filo-Corona
o Formation of fused cells/ syncytial cells → RSV
All are enveloped, except CPR Calici-Picorna-Reo
o Vacuoles in cytoplasm/ foamy degeneration → HPV
All replicates in cytoplasm, except OR Ortho-Retro
o Swelling and Clumping → Adenovirus
Smallest RNA Picorna
HEMADSORPTION
Largest RNA Paramyxo
- Observe for virus with little or no CPE
Arthropod-borne Arbo-Flavi-Bunya-Toga
- Example: Influenzae, Parainfluenzae, Mumps
- Ability to stick to mammalian RBCs
 DNA VIRUS NS1 Antigen Test → detect current
ADENOVIRIDAE Adenovirus infection
HEPADNAVIRIDAE Hepatitis St. Louis Encephalitis Culex Mosquitos (Night)
Parvo B19 → 5th disease; Erythema infectiosum Zika Virus Aedes Aegypti
PARVOVIRIDAE
Erythrovirus Aedes albopictus
BK or PVH 1 Infant: microcephaly
POLYOMAVIRIDAE JC or PVH 2 Adults: GBS
MC West Nile Virus Culex
Variola or Orthopox Fever and Encephalitis
Alastrim Hepatitis C Hepatocellular Carcinoma
POXVIRIDAE Vaccinia Chronic Liver Disease
Molloscum contagiosum MOT: Blood; sex; parenteral
Orf Virus
PAPILLOMAVIRIDAE HPV Anti-HCV → not protective
ASFAVIRIDAE African Swine Flu ALT → surrogate marker
Alpha Bunyavirus Arthropod borne
- HHV 1 Mosquito, ticks, sandflies
- HHV 2 Rift valley, La crosse, California, Crimean
- HHV 3 (VZV) congo
Beta Hanta virus Rodent-borne
HERPESVIRIDAE - HHV5 (CMV) BUNYAVIRIDAE HFRS (Hemorrhagic Fever Renal
- HHV 6 (6th Dse) Syndrome)
- HHV 7 Muerto / Sin (deer mouse)
Gamma New York (White footed mouse)
- HHV4 (EBV) Bayou (Rice rat)
- HHV8 Black creek Canal (Cotton rat)
RNA VIRUS Non-Arthropod Borne
Arthropod-borne New World Arena Virus Hemorrhagic Fever
Alpha Virus Equine encephalitis Junin, Machupo,
Thru mosquitos Tacaribe, Guanarito
Chikungunya Myositis and arthritis ARENAVIRIDAE Old World Arena Virus Aseptic Meningitis
TOGAVIRIDAE Aedes aegypti Lymphocytic Hemorrhagic Fever
Rubella Virus German measles Choriomeningitis
Low grade fever; few rash; 3-day Lassa Fever Virus
Forrcheimer spots ASTROVIRIDAE Astrovirus Gastroenteritis
Yellow Fever Hemorrhagic Fever Norovirus/Norwalk Diarrhea in Norwalk
FLAVIVIRIDAE Dengue Fever Aedes aegypti (Day-bite) CALICIVIRIDAE Sapovirus Diarrhea in infants, elderly and young
E protein children
FILOVIRIDAE Marburg Affrican Green Monkey
 Shepherd’s Crook Morphology Serotypes: Respiratory secretions
Ebola Virus/ Zaire/ Emerging virus in Africa 1. Influenza A = animals Co-bacterial infection due to NA (MRSA)
Reston/ Tai Forest/ Direct contact with infected animal or 2. Influenza B = humans
Ivory Coast human 3. Influenza C = Humans: H1-H3; N1-N2
BSL4 ORTHOMYXOVIRIDAE humans, subclinical
Hepatitis E Hepatits E
HEPEVIRIDAE
Fecal-Oral HA → Hemag.
Rotavirus Most common cause of Infantile Attachment
REOVIRIDAE NA → Neura. Release
Colorado Tick Fever diarrhea
Rabies Virus Saliva frothing with painful spasm Mumps or Rubulavirus Parotid glands
Lyssa Virus Coma or death (no vaccine given; Orchitis (infertility)
RHABDOVIRIDAE
aritificial passive vaccine) Parainfluenzae Virus Common cold syndrome
Negri bodies (brain tissues) Parainfluenzae 3 Pneumonia, bronchiolitis
Oncoviruses Flower nuclei in T-cells Parainfluenzae serotype Croup/ acute tracheolaryngobronchitis
RETROVIRIDAE HTLV 1,2,5 1
(WITH REVERSE Lentivirus HIV Ag: p24, gp41, gp120, gp160 RSV F protein
TRANSCRIPTASE) HIV Screen: ELISA 2x PARAMYXOVIRIDAE Common cause of viral pneumonia
Confirm: Western blot; RhiVda among children (5 y.o)
Rhinovirus Major cause of Common Colds Measles or Morbilivirus Macupapular rash: Koplik’s Spots
Acid Labile (pH <6) CPE: Giant Cells SSPE
Enterovirus Most common cause of aseptic High-grade fever, many rash, 7-day
meningitis or inflammation of brain Metapneumovirus Winter epidemics of bronchiolitis and
parenchyma pneumonia
Nipah Virus Febrile encephalitis
Respiratory or gastrointestinal Corona virus SARS-COV1 : Civet; Guangdong
Poliovirus (1-3) Paralytic Poliomyelitis (flaccid) MERS-COV : Camel; Middle East
Infantile Paralysis Testing: SARS-COV2: Pangolin; Wuhan
Infect Anterior horn of spinal cord 1. Direct: Microscopy/
PICORNAVIRIDAE Coxsackie A (1-23) Herpangina (painful mouth infection) PCR
Hand and footh and mouth Dse 2. Indirect: serological PCR Analysis Samples:
CORONAVIRIDAE
Coxsackie B (1-6) Epidemic myalgia NPS (best), OPS, Saliva
Pleurodynia (painful upon breathing)/ Handling:
devil’s grip A. Non-propagative: Antigen Test → Infection NOW
Bornholm Dse diagnostic assays; BSL 2 Antibody Test → PAST Infection
ECHO Virus (1-32) Infantile diarrhea and aseptic meningitis B. Propagative: viral NAAT → Gold Standard; NOW
Enterovirus 72/ Infectious/ epidemic hepatitis culture; BSL 3
Hepatitis A virus No chronic state
Fecal-oral
Parechovirus (1-4) Summer diarrhea