Midterms Wizz G.
MicroPara Laboratory 1—I
The Use of Phys & Chem Methods for
Microbial Control • Aldehydes
Broad spectrum and toxic. For
May either be Disinfection (temporary and not
endospores.
totally killing) or Sterilization (longer duration
and killing all MO)
• Halogens
Has chlorine (hypochlorite) for hard
surface, and iodine for skin.
Sterilization: Physical • Alcohol
Broad spectrum. May be Isopropyl or
• Heat Ethyl.
o Moist Heat
Uses autoclave or pressure • Quaternary Ammonium Compounds
cooker. By coagulating proteins For example; Cetrimide – antimicrobial
using steam. The lesser moisture but not for spores, Mycobacterium, and
the higher the temp. 121° / 134° increases Pseudomonas growth. For
C for 15 / 13 mins, respectively. skin, but not theater (since it has
For surgery. detergent properties).
o Dry Heat
Uses hot air oven on 160 ° –
Important Terms:
180 ° for 20 – 60 mins. For
ointment and Lab app. SDA – Sabouraud Dextrose Agar
Formula for Percent Reduction:
• Radiation
Uses free radicals to disrupt intercellular
(IC) molecules and DNA. Surgical
gloves, syringe and needles, infusion
sets and prostheses.
70% Alc. is the most effective concentration.
• Filtration Autoclaving is the best methos of sterilization.
Uses membrane filters w/ 0.22 – 0.45
µm pore size. Seitz (asbestos), Millipore
(cellulose membrane), Gracodol and
Collodion (nutricellulose), Chamberland
(unglazed porcelain), and Berkefield Antimicrobial Susceptibility Testing (Kirby –
(diatomaceous earth). Technically not Bauer Technique)
sterilization since it doesn’t kill viruses.
Chosen above other methods due to its efficacy
and simplicity.
Sterilization: Chemical
In this technique, standard amt of antibiotic is
• Ethylene Oxide placed on Mueller-Hinton Agar (MHA) w/
Gas Sterilizers. Alkylating agents that standard amt of inoculum (1.5x108 cells/ml).
bind to protein and nucleic acid. Blocks Undergoes 24 hrs incubation.
DNA replication (DNAr). Slow acting.
Disinfection: Chemical Factors that may affect results:
• Phenolics • suitability and pH of the medium
Disinfection of inanimate objects such • amt of inoculum
as floors. • thickness of agar
• diffusion of antibiotics on the disc
• Chlorhexidine • time
Narrow spectrum and non-toxic. Used • incubation temp
for surgical disinfection.
Midterms Wizz G.
MicroPara Laboratory 1—I
2 Different Methods: Results:
o Acid = Yellow, Alkaline =
• Direct Colony Suspension Method Red/Orange
Key points: o Alk slant, alk butt (no sugar)
o Inoculum is prepared by o Alk stant, acid butt (only
suspension of isolated colonies glucose, no sucrose/lactose)
in agar plate (18 - 24 hrs). o Acid slant, acid butt (glucose &
o Adjust suspension w/ lactose [or sucrose in TSI]
McFarland standard. fermented)
• Growth Method or Lag Phase Method • Oxidative-Fermentative (OF) Test of
Key points: Carbohydrates (Hugh and Liefson’s)
o 3 – 5 isolated colonies w/ same Key points:
morphology. o Gram-negative bacteria that
o Incubate broth at 37° for 2hrs. undergo aerobic respiration w/
o Adjust to 0.5 McFarland weak acids got thru: Krebs cycle
standard. & Entner Douodoroff
(glygolysis)
o Increased conc is detected by
Important Terms: bromothymol blue indicator
After disc implant, invert plates and into o Dipotassium buffer for acid
detection
incubator (35 – 37° for 16 – 18 hrs).
Results:
If discreet colonies are seen, measure visible o Fermentative results: yellow
colony-free zone only. from green
o Oxidative: yellow near on top
Sizes of Zones are interpreted using M100 tables
only
2A to 2I.
o Negative: no change to yellow
MO are most sensitive during Log Phase.
• Carbohydrate Utilization Test (Cystic
Tryptic Agar-Based)
Key points:
o Semisolid w/ essential nutrients
Test for Carbohydrate Fermentation for fastidious organisms
o Has phenol red indicator
Fermentation is an anaerobic degradation of
o For differentiation of gram-
carbs. Carbohydrate fermentation is invaluable
negative diplococci
in determining sugar utilization in some bacteria.
Results:
o Fermentation: red to yellow
o Negative: neutral orange
Three basic requirements: o Neutral orange is due to the
• carbohydrate of interest deamination of peptone if no
• pH indicator carbs source
• inverted tube (Durham tube)
Three tests: Important Terms:
• Sugar Fermentation TSIA- Triple Sugar Iron Agar
Key points:
o Tryptichydrolysate of casein OF- Oxidative Fermentative
(source of carbon and nitrogen) Fermentation is anaerobic, while oxidation is
o NaCl (osmotic stabilizer) aerobic which leads to oxide formation.
o Phenol red (indicator)
o Carbohydrate to be tested (0.5%
- 1% conc.)
Midterms Wizz G.
MicroPara Laboratory 1—I
IMViC Tests Results:
Stands for Indole, Methyl Red, Voges o Positive: Green to blue
Proskauer, and Citrate Utilization test.
Most popular tests include E. coli (IMViC ++--)
Bacteriologic Examination of Water
and Enterobacter aerogenes (IMViC --++)
Potability of H2O must pass the physical,
chemical, and bacteriologic testing.
A. Indole Production
Bacteriologic testing screens for coliforms and
Key Points:
heterotrophic plates.
o Indole, product from amino acid
degradation Coliforms are bacteria that have originated from
o Bacteria w/ tryptophanase can GI tract.
cleave thru tryptophan and
produce: indole, pyruvic acid, Colon bacillus are the BEST index for fecal
and ammonia contaminated water.
o Uses Su;fide Indole Motility Some survive 44° (thermotolerant)
(SIM) or Motility Indole
Lysine (MIL) tubes H2O is not potable if >500 CFU/ml
o Uses 6 – 8 gtts of Kovac’s pr
Erlich’s reagent
Waterborne dses:
Results:
• hepa A
o Positive: Pink or red at the
surface of agar. • amoebiasis
• cholera
B. Methyl Red Test • typhoid fever
Key Points: • dysentery
o pH below 4.4 is the breakpoint Types of Coliforms:
of methyl red indicator
o MRVP medium (Methyl Red • total
& Voges-Proskauer) • fecal
• thermotolerant
Results:
o Positive: Red color in surface of
medium
Test for Coliform Group
C. Voges-Proskauer Test 1. Presumptive Test for Total Coliform
Key Points: Organisms
o Detect the formation of o <100 ml H2O and within 2hrs
acetylmethylcarbinol (acetoin) o Incubate for 24hrs for gas. If
o MRVP medium none then 24 more. No gases
o 6-8 gtts 5% α-naphtol, & 4 after 48hrs is NEGATIVE.
drops of 40% potassium o If gas after 24hrs then proceed
hydroxide to confirmed test.
Results:
2. Confirmed Test for Fecal Coliform
o Positive: Red color in surface Organisms
o Transfer 1-2 gtts of E.C
D. Citrate Utilization Test (Escherichia coli) medium
Key Points: broth and BGLB (Brilliant
o Determine if bacterium can use Green Bile Lactose Broth).
citrate as source of carbon. o Incubate for 24-48hrs to observe
o Simmon Citrate Agar coliforms in sample
Midterms Wizz G.
MicroPara Laboratory 1—I
o To confirm thermotolerant Classification by Color:
coliforms then incubate for 24
and observe for gas formation. • Hyaline: light-colored
• Dematiaceous: dark-colored
3. Completed Tests Types of Infection:
o Isolate from a plated media.
Proceed to study. • Endothrix (inside hair shaft)
• Ectothrix (outside hair shaft)
Heterotrophic Plate Count (Pour Plate
Method) Procedure:
o Uses 1ml H2O • Material
o Choose plate w/ 30-300 a. Skin
colonies Cleanse w/ 70% alcohol. If
o Don’t include TNTC ringworm is present, outer red
o Report in CFU/ml portion is scraped
o CFU- Colony Forming Unit b. Hair
Broken and scaly hairs. By the
roots w/ tweezers.
Potassium Hydroxide (KOH) Mount for the c. Nail
Examination of Mycoses Clean w/ 70% alcohol. Use
blade and scrape outer layer.
Fungi is hard to distinguish from other debris d. Urine & body fluids
and must be stained. Concentrated by centrifuge.
Potassium hydroxide is used to dissolve other Only sediments are examined.
debris leaving fungi. • Add 1-2 gtts of 10% KOH
• Let stand for 10 – 30 mins
NaOH is the best sub for KOH. • If hard, gently heat 2-3 times
• Flatten w/ finger
• Examine thru EM
Fungi can be mistaken as:
Results:
• RBC
• Plant hair • No debris.
• Pollen grain • Fungi can be classified
• Algae
• Fiber
Keratinous materials:
• Skin
• Hair
• Nails
Fungi infects:
• Epidermis (superficial mycoses)
• Dermis (cutaneous mycoses)
Structures of Fungi:
• Hyphae “spaghetti”
• Spores “meatballs”
Classification of Hyphae:
• Septate (w/ cross walls)
• Aseptate (w/o or coenocytic)
