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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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