Chemical methods

a. Ethylene oxide- most commonly used chemical sterilant, used in gaseous form for sterilizing heat sensitive
object indicator:
b. Formaldehyde vapor and vapor phase hydrogen peroxide- used to sterilize heap filter
c. Glutaraldehyde 2% sporicidal, kills spores in 3-10 hours, for medical equipment
d. Periacetic acid- against all vegetative microorganism as well as the bacterial and fungal spores

B. DISINFECTION

Physical Method

1. Pasteurization -63 degc 30 min batch/ vat pasteurization; 72 degc 15 sec increase temp short time (Flash
Method); 140 degc 3 sec- increase temp short time
2. Boiling – 100 degc 15-30 min for surgical instrument
3. UV- non-ionizing radiation using UV light

Chemical Method

1. Antiseptic-chemical germicide use on the skin or tissue and not to be substituted for a disinfectant

Alcohols- 70-80 (60-90%mahon)

Iodophors- BEST antiseptic composed of iodine + detergent ( natural polymer)
Chlorhexidine- whole body disinfection of px undergo surgery.
Hexachorophene- 3% available only by prescription

2. Disinfectants- for non-living things.

Halogens (Chlorine, Iodine, Fluorine)
Heavy Metals
Aldehydes
QUATS –Quaternary ammonium compounds e.i. Benzalkonium Chloride and Zephiran
Pseudomonas aeruginosa grown in the ammonium acetate media is RESISTANT to QUATS.
Phenolics

EXAMPLES OF BACTERIA, WHICH REMAINS ACTIVE IN A DRY ENVIRONMENT

N. gonorrhoeae- 1 hr
M. tuberculosis- several months
Bacillus and Clostridium- 10 years.

SPECIMEN PROCESSING

A. Transport- ideally, specimens should be transported to the lab within 2 hours of collection.
B. Preservation
-Boric acid to maintain a colony count accurately
-SPS 0.025% prevents clotting. EDTA and Citrate be NEVER use. Heparin is for viral cultures, inhibits gm+
and yeast.
C. Storage
-CSF is at RT, NEVER refrigerate.
-Urine, stool, viral specimen, sputum, swab are REF temp
-Serum for sero -20 deg C for 1 week
-tissues and anaerobic cultures for long storage at -70 deg C.
WAYS TO FACILITATE ANAEROBIC CULTIVATION

-Boiling of medium to remove O2

-Use of GAS PACK JAR with palladium catalyst; indicator METHYLENE BLUE.
-anaerobic cultures are incubated at 35-37 deg c.

CLINICAL SPECIMEN

1. Blood
-most common anticoag is SPS 0.025% to prevent clotting.
-dilution factor blood to anticoag ratio is 1:10
-adult is 10 ml (5 ml for aerobic, 5 ml for anaerobic)
-Children (1-5 ml)
-routine blood culture is 7 days
-Brucellosis 3-4 weeks, leptospirosis 8 weeks.

2. CSF
-Collected in 3-4 tubes. Tube #2 is for micro, gm and culture, if fourth tube is collected, use the fourth
tube.
-examine immediately. If not, hold at RT or @ 37 deg C no longer than 1 hr.
-Never Refrigerate.
-centrifuge, use sediments for smears and culture.
-common pathogens are N. meningitidis, H. influenza,S. pneumoniae

3. Throat and Nasopharyngeal swabs

-Most abundant normal flora: S. viridans
-Most pathogenic: S. pyogenes
-Culture in Todd Hewitt Broth for Fluorescent Microscopy of β strep
-Nasophangeal swab is the specimen of choice to detect the Carrier state of N. meningitidis, presence of
B. pertussis and H. influenza.

4. Sputum
-Often contaminated with normal flora
-Note the number of Squamous epithelial cell/LPF and PMNs.
- Accept if >25 PMNs, <10 SEC. REJECT if not.
-Gram stain is performed on all specimen
-Collected ideally on AM when it’s most concentrated through deep cough (3 samples on alternative days,
DOH 2 samples)

5. Urine
-Mid-stream clean catch is the specimen of choice, Catheter patients.
-Must be preserve or refrigerated if not processed.
-Boric acid maintain accurate count.
-Major cause of UTI is E. coli
-UTI among young and sexually active women is S. saprophyticus.
-MAC and BAP are suitable combination
-Colony count should be perform in all samples.
 6. Stool
-Detection of enteric pathogen
-sample not process within two hours of collection should be placed on transport media such as Cary
Blair.
-This specimen is routinely screen for Salmonella, Shigella, and Capmpylobacter
-Directly plated on MAC, EMB, SSA, HEA

7. Genital Tract
-Dectects presence of N. gonorrhoeae, G. vaginalis and C. trachomatis for cervicitis.

WAYS OF STUDYING BACTERIA

 Study of morphology/appearance
-Size, shape, motility, arrangement, and staining characteristics.
-Motile check is presence of flagella or just Brownian movement.

 Staining
-artificially coloring the organism
-to abserve the appearance of the bacteria
-to differentiate one organism from another.
-to reveal the chemical nature of bacteria

 Staining Techniques
-Simple/Direct: use 1 dye
-Idirect/Negative: stains the background (India ink, Borris method and Nigrosin)

Special Staining stains special part of the cell.

Capsular Stain: Hiss, Anthonys, Tyler, Muir

Spore Stains: Dorners, Schaeffer Fulton, Wirts and Conklin
Flagellar Stain: Grays, Fisher and Conn, Leifson
Metachromatic Granules: Alberts, Methylene blue,Ponder
Nucleic acid: Fuelgen
Polar Bodies: Wayson
Spirochetes: Levatidi,Warthin Starry, Fontana-tribondeau
Cell wall: Dyar

Differential Staining- use to differentiate organisms from one another. Two most common are Gram Stain
and Acid Fast Stain.

A. Gram stain

Purpose Reagent Gram positive Gram negative

Primary stain/ initial Crystal violet violet violet
stain
Mordant Grams iodine violet violet
Decolorizer 95% ethyl or acetone violet colorless
Counterstain or safranin Violet red
Secondary stain
 Huckers modification- grams for fungi; add ammonium oxalate + crystal violet

 Mycobacterium
 Corynebacterium
 Clostridium
 Bacillus
 Erysipelothrix
 Lactobacillus
 Listeria

3. Higher form of organism like Actinomyces, Streptomyces, yeast and molds are gram (+)
4. All spiral organism are reported as gram (-)
 Not gram strained are Rickettsiate; Chlamydia, Mycoplasma (wall less), spirochetes’

Reasons why grams (+) becomes gram (-) Reasons why gram(-) becomes gram (+)

Over decolorization Under decolorization

Use of grams and iodine (alkaline) Thick bacterial smears

a. Acid fast stain

 To distinguish AF from NAF organism
 ACID FAST ORGANISM- are organism that are very hard to stained they are difficult to
decolorize due to MYCLOTIC ACID/ HYDROXYMETHOXY ACID that envelopes the bacteria
 Rule: all bacteria are not acid fast except: MYCOBACTERIUM, slightly acid fast is NOCARDIA
Ways to facilitate acid fast staining
 Steaming process
 Increasing concentration of phenol and basic fuchsin
 Prolonging contact of stain with the msterial
 Addition of wetting agents (tergitol) prior to the stain solution
Purpose Ziehl Neelsen/ Hot method Kinyoun / Cold method
Primary 3 grams Carbol fucshin + 5% phenol 4 grams CF + 9% phenol
Mordant Heat/ streaming tergitol
Decolorizer Acid alcohol (HCL + ETHYL) Acid Alcohol (HCL + Ethyl)
Counterstain Methylene blue Malachite green
Result Red in blue background

 Size of AFB smear- 2x3 cm

 AF organism in tissues is best stained using KINYOUN
 AFB smear can be fix using slide warmer set at 65-67 deg C for 1-2 hours or 80 deg C for 15
mins on electric hot plate.

OTHER METHODS OF ACID FAST STAINING

 Pappenheims to differentiate M. smegmatis from M. tuberculosis (red)
 Baumgartens to differentiate M. leprae (red) from M. tuberculosis
 Fite Faraco’s M. leprae Hematoxylin as counter stain.
STUDY OF CULTURAL/COLONIAL CHARACTERISTICS
1. According to Physical State/Consistency
 Liquid: No solidifying agent (0% agar) Ex Broth, APW, BHI
 Semi-Solid: 0.5-1% agar. Ex SIM
Agar- usually derive from red algae, solidifies at 40-50 deg C and melts at 80-90 deg C
Cooling temperature for distribution of media to plate 55-60 deg C.
 Solid
Liquefiable contains 2-3% agar (EMB, MC Conkey)
Non-liquefiable: Rice medium for fungi
 Biphasic Media: both liquid and solid (Castaᾖeda medium)
2. According to Composition
a) Synthetic/Defined- all component are known to the user, for research (BG-11 for Cyanobacteria)
b) Non-synthetic/Complex- composed of some unknown substance (peptone,meat) useful for
bacterial isolation. Ex: EMB, PSB
c) Tissue Culture/Living Cells- for viral culture- Cytopathic Effect.
 HeLA cells: cervical cancer
 McCoy Cells: Chlamydia
 Chick Embryo: Rickettsia and Virus
 A549: lung carcinoma
 Hep2 cells: laryngeal cancer
 Vero cells: from African green monkey kidney cells
 KB: nasopharyngeal cancer
3. According to Use
a. General isolation media- contains only the necessary nutrition to support bacterial growth, for
routine cultivation of nutrient. Ex Nutrient broth/ Nutrient Agar
b. Enrichment Media- Increase growth, Increase bacterial yield. Ex APW, Tetrathionate for
Chlamydia
c. Enriched Media- for fastidious organisms. Ex BAP and CHOC
d. Selective Media- promotes the growth of the desired organism while inhibiting the growth of
others. Ex McConkey.