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Midterms-LAB- Complete
Medical Technology (Far Eastern University)
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INOCULATION TECHNIQUE
Isolate microorganism on culture media
To plot/streak microorganism on culture media
Inoculating wires
Made up of either nichrome/aluminum
Sterilization: to eliminate unwanted organism using dry heat
Types:
1. Inoculating needle – for tube media
2. Inoculating loop – transfer microorganism on to solid/plated media
3. Calibrated loop – the specimen that will be transferred has a specific volume (0.01mL) from liquid type ~ usually
urine
4. Bent wire – for fungal specimen
Inoculating Technique in regards to the type of culture media
1. Tube Media
a. Liquid
Brain-Heart Infusion Broth
Inoculating Loop/Needle is used
Do not transfer microorganism directly in broth
i. Slant the tube slightly and place it on the side of the tube, without touching the broth, then mix
b. Semi-solid
Sulfide Indole Motility
Inoculating Loop/Needle is used
i. Stab method ~ point of entry is also point of exit
ii. Avoid jerking action
c. Solid
Simmon Citrate Agar
Inoculating Loop/Needle for Slanted
Inoculating Needle for Butt & Slant
i. Stab & Streak Method
2. Plated Media
a. Interrupted Streak Method
Inoculating Loop/Needle is used
i. Make an imaginary line on the medium, then start at ½ cm from plate.
ii. Rotate at 180º to make secondary streak
b. Overlap Streak method
Inoculating loop is used
i. Make a point of inoculation then streak descending
ii. Inoculate
iii. Do the secondary and tertiary streak
Growth in first streak: Light growth
Growth in first and second streak: Moderate growth
Growth in first, second and third streak: Heavy growth
c. Multiple Interrupted
Inoculating loop is used
i. Streak in place then touch the previous streak
d. Multiple Inoculation
Inoculating loop is used
Different bacteria can be plated
Difficult to establish isolated colonies
e. Radial Streak
Inoculating loop is used
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i. Make a point of inoculation then make vertical streaks
ii. Streaks must not touch each other
f. Pour plate
Specimen: Stool, Milk and Water
Serial Dilution: decrease population size using a diluent (Normal Saline Solution, Distilled Water)
i. Prepare 4 test tubes with 9 mL diluent (NSS) each
ii. Get 1 mL of the specimen and transfer it to a first tube.
iii. Get 1mL from the first tube then transfer it to a second tube and so on
iv. Prepare 10-12 mL of agar then transfer to a plate that is about to solidify then mix
BACTERIAL CHALLENGE TEST
Materials:
Culture Media Chemical agents:
Alcohol Lamp Hypochlorite Solution
Inoculating Loop Liquid Soap
Labeling Materials Lysol
Culture of Bacteria 70% Alcohol
Procedure:
1. Divide plated culture media into 4 quadrants and label each quadrant with the time: 30 secs, 1 min, 2 min, and control
2. In a sterile test tube, add 0.5 mL of chemical agent
3. Using a sterile inoculating loop, transfer culture of bacteria into chemical agent and start the time once the contact of
organisms to the chemical agent has started
4. Exactly on the time indicated, transfer and streak on labeled culture media
5. Incubate at 35ºC for 18-24 hours
Mac – Escherichia coli
BAP – Staphylococcus aureus
*Any growth confirms insufficient effectiveness of chemical agent
Factors that influence the Activity of Chemical Agents
A. Types of Microorganism
B. Temperature and pH
C. Number of organisms – higher number of organism, lesser effectiveness
D. Concentration of chemical
E. Amount of organic substance – mucous, blood, sputum (more amount, less effective)
F. Nature of surface to be disinfected
G. Length of contact time
H. Type of water used
PREPARATION OF MCFARLAND STANDARD
Solution is a combination of BaCl 3 and H2SO4 to for a precipitate
Will have a specified optical turbidity
Purpose:
Used as a standard in preparing pure inoculum for AST
Used to study bacterial growth curve
Used to determine an increase or decrease of the concentration of vaccine
Research purposes
Appropriate volume of reagent
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1 2 3 4 5 6 7 8 9 10
1.175% BaCl3 0.01 0.02 0.03 0.04 0.05 0.04 0.03 0.02 0.01 0.1
1% H2SO4 9.99 9.98 9.97 9.96 9.95 9.96 9.97 9.98 9.99 9.9
Approx. Density 3 6 9 12 15 18 21 24 27 30
( x 108 CFU/mL)
ANTI-MICROBIAL SUSCEPTIBILITY TEST
(Kirby Bauer/Disc Diffusion Method)
Materials:
Alcohol Lamp MHA
Inoculating Needle Sterile Distilled Water/TSB/BHIB/NSS
Sterile Applicator Swab Ruler or Micro caliper
Forceps Antibiotics
Labelling Materials
Specimen:
Plated Culture [Staph spp., Gram (-) bacteria]
Procedure:
1. Preparation of pure inoculum
Get 3-5 colonies then transfer to the broth
2. Standardization of pure inoculum
0.5 McFarland
If McFarland is more turbid than inoculum, add colonies
If inoculum is more turbid, add sterile distilled water
3. Streak inoculum using a sterile cotton swab onto MHA
2-3 strokes
4. Apply antibiotic discs on streaked MHA
5. Incubate at 35ºC for 24 hours
6. Measure zone of inhibition and interpret
Gram Negative Antibiotics
ANTIBIOTICS ABBREVIATION RESISTANT INTERMEDIATE SUSCEPTIBLE
Amoxicillin AX <19 -- >20
Ampicillin AM <13 14-16 >17
Cabenicillin PY/CB <19 20-22 >23
Cefuroxime CXM <14 15-17 >18
Cephalothin KF <14 15-17 >18
Chloramphenicol C <12 13-17 >18
Ciprofloxacin CIP <13 14-17 >18
Ertapenem ETP <12 13-15 >16
Gentamycin CN/GM <13 14-17 >18
Kanamycin K <13 14-17 >18
Nitrofurantoin NI <14 15-16 >17
Ticarcillin TIC <14 15-19 >20
Tetracycline TE <14 15-18 >19
*Nitrofurantoin – inhibits DNA, RNA, Protein and Cell Wall Synthesis
Gram Positive Antibiotics
ANTIBIOTICS ABBREVIATION RESISTANT INTERMEDIATE SUSCEPTIBLE
Araoxicillin/Cluvanic Acid AMC <19 -- >20
Aztreonam ATM <15 16-21 >22
Bacitracin B <8 9-12 >13
Cephalothin KF <14 15-17 >18
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Ciprofloxacin CIP <13 14-17 >18
Clindamycin DA <13 14-17 >18
Erythromycin E <13 14-16 >17
Nalidixic Acid NA <13 14-18 >19
Novobiocin NV <14 15-16 >17
Oxacillin OX <17 -- >18
Penicillin G P <28 -- >29
Streptomycin S <13 14-17 >18
Vancomycin VA <14 -- >15
Tetracycline TE <13 14-17 >18
*Novobiocin – Inhibits Protein Synthesis
*Cefoxitin – Resistant: </=21
Susceptible: >/=22
CLINICAL SPECIMEN COLLECTION FOR BLOOD CULTURE
Clinical Specimen – collected from patients suspected with disease
Blood Culture – must be done before antibiotics are given
Materials:
Venipuncture Set
10 mL Thioglycolate
Bacteria:
Escherichia coli
Staphylococcus aureus
Blood Culture Indications:
Indication Duration of Culture Culture Medium
Bacteremia/Septicemia Trypticase Soy Broth
Endocarditis 5-10 days @ 35ºC Brain-Heart Infusion Broth
Bacteria of unknown origin Thioglycolate
Brucella Broth
Brucellosis 3-4 weeks Wisconsin Medium
Castaneda medium
Fletcher’s medium
Leptospirosis 5-6 weeks
EMJH
IDENTIFICATION OF STAPHYLOCOCCUS SPP.
Turbid – gram (-) bacilli
Jelly-like coagulum – Staph. Aureus
A. Morphological Characteristics
a. Colony appearance – pinhead; Medium to Large colonies
b. Gram Stain
Culture Media: BAP
Gram (+) cocci in clusters ~ violet/purple
B. Biochemical Characteristics
a. Catalase Test
Reagent: 3% H2O2
(+) Effervescence
b. Coagulase Test (Best test to differentiate Staphylococci)
Tube (unbound)
0.5 mL plasma + bacteria
35ºC for 4 hours
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Slide (bound)
Plasma + bacteria
(+) Clot/fibrin formation = S. aureus
(-) No clot = CONS
Use of citrated plasma (Ideally Rabbit’s Plasma)
c. Mannitol Fermentation
Subculture on MSA
35ºC for 4 hours
(+) Yellow = S. aureus
(-) Pink = CONS
d. Novobiocin Susceptibility
Susceptible = All Staph
Resistant = S. saprophyiticus
e. Bacitracin Susceptibility
Susceptible = All Staph
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