Video presentation of

Procedures in Bacteriology
laboratory
Prepared by : Precious Mae Ballesteros RMT,
MLS ASCPi
AUTOCLAVING OF GLASSWARES
HOW TO PREPARE CULTURE MEDIA?
A. AGAR PLATE B. PROCEDURE
Materials: petri dish, graduated
1. Prepare all the materials needed
cylinder, erlenmeyer flask,
2. Prepare 500 ml of nutrient agar mixture ( D. H20) with
stirring rod, weighing scale , agar
the following :
powder ( NUTRIENT AGAR) ,
spatula , distilled water
 How to compute?

Nutrient Agar Follow the ratio and proportion:

23grams of Nutrient agar powder to 23g x 500 / 1000 = 11.5 g of

1 Liter of Distilled water agar needed

Prepare : 500ml of nutrient agar

 Prepare 650 ml of Nutrient agar

Try to compute 32g of Nutrient agar to 1L of Distilled

water
How to
prepare
blood agar
plate?
How to make
chocolate agar?
HOW TO PREPARE CULTURE BROTH?

END AT 5 MIN MARK

How to prepare
agar slant?
BACTERIAL
INOCULATION
TECHNIQUES
SAMPLE
INOCULATION

AND SMEAR
PREPARATION
STREAK
PLATE
METHOD
Describing
colony
morphology
Smooth glistening convex
colonies
Rough

Actinobacteria
Wrinkled

B. psuedomallei
Dry
and
powder
y
Bacterial
smear
ANTIMICROBIAL
SUSCEPTIBILITY
TESTING
 1. TO DETERMINE THE CAPACITY
OF THE BACTERIAL ISOLATE
TO CAUSE DISEASE
2. TO IDENTIFY THE
SUSCEPTIBILITY PATTERNS
PURPOSE OF THE BACTERIAL ISOLATE
TO THE ANTIMICROBIAL
AGENTS
3. TO ASCERTAIN THE
AVAILABILITY OF THE
STANDARD SUSCEPTIBILITY
METHODS
MUELLER
HINTON AGAR
- Standard susceptibility medium
for non fastidious bacteria
- Recommended medium for
susceptibility testing because it
has reduced concentration of
sulfonamide, trimethoprim and
tetracycline inhibitor
- Composed of beef infusion, nucleic acids, vitamins ,
casein hydrolysate, ( casein neutralizes fatty acid) and
agar
- MH Broth with 2 % NaCl is used to improve the detection
of oxacillin resistant staphylococci
- An MH broth with lysed horse blood or sheep blood is
utilized for testing the susceptibility for streptococci,
Neisseria meningitidis and other fastidious organism
terminology

1. Breakpoint (cut- off) - refers to the concentration of an antimicrobial agent

that coincides with a susceptible or intermediate MIC breakpoint for a
particular drug.
2. Minimum bactericidal concentration ( MBC) - lowest concentration of the
antimicrobial agent that is needed to kill the bacteria
3. Minimum inhibitory concentration - (MIC) - lowest concentration of the
antimicrobial agent that inhibits the bacterial growth
4. Minimum lethal concentration (MLC) - lowest concentration of the
antimicrobial agent that kills the bacterial growth when subcultured in a
fresh medium
5. Paradoxic (eagle ) effect - defined as the decrease bactericidal activity at a
higher drug concentration

6. Skipped wells - involve the growth at higher concentrations and no growth at 1

or more of the lower concentrations

- May indicated contamination , improper incubation improper concentration of

the antimicrobials and the presence of an unusual resistant isolate

7. Tolerance - ability of an microorganism to be inhibited by a drug which is

normally bactericidal ; defined with an MBC: MIC ratio of >/= 32

8. Trailing growth - involves heavy bacterial growth at lower concentrations

that is followed by one or more wells that show a greatly reduced growth in
the form of a small button or a light haze.
Broth dilution method

- Involves in challenging the organism of Procedure :

interest with antimicrobial agents in a
broth environment ( MH BROTH Specific amount of antibiotic is prepared in a
- Used to determine MIC and MLC decreasing concentration in broth by a serial
- Serial two fold dilution concentration dilution technique in which a standard amount
( preferred) of the test organism is then inoculated
- Standard inoculum size is 5x 10 ^5 CFU/
ml

Result: absence of turbidity


Broth macrodilution
- MH broth
- Broth volume : >/= 1 ml
- Final standard inoculun : 5x10^5 CFU/ml
- Incubation time : 16-24hrs at 35C
- Testing for fastidious bacteria and
identifying MBC end point
- Disadvantage : impractical when used to
test several antimicrobial agents or isolates
Broth microdilution
- MH broth
- Broth volume : .05 ml ot 1 ml
- Inoculum size : 5x10^5 CFU/.1ml
- Incubation time ; 16-24hrs at 35C
and 48 hrs at 35C anaerobes
Advantage : for testing different
antimicrobials 10-15 at the same time
against single isolate
Agar dilution method

Reference method for testing anaerobes and N.

gonorrhoeae

- Frequently used in research

- BRUCELLA- LAKED SHEEP BLOOD
( Wadsworth method) medium used for
anaerobes
- Susceptibility testing is MHA
- 5% defibrinated sheep’s blood is added
into the MHA for fastidious bacterias
Disk Diffusion ( Kirby bauer test)

Principle : The larger the zone of inhibition, the

lower the MIC; the zone of inhibition is inversely
related to MIC

Standard inoculum size : 1.5x10^8 CFU/ml

Disadvatntage : not recommended for testing

slow growers like mycobacteria and anaerobes
McFarland Standard

.5% McFarland turbidity standard ( Barium

sulfate suspension

- Equivalent to 1.5x10^8 CFU/ml

Preparation of Pure culture for Disk Diffusion
Susceptibility testing

1. Pure inoculum is obtained by selecting 4-

5 colonies of the same morphotype then
inoculate into TSB and incubate at 35C for
18 to 24 hrs
2. The bacterial suspension and McFarland
solution are compared by matching the
turbidity of the tubes against a dark
background
3. If the bacterial suspension do not match
with the standard’s turbidity, the
suspension may be diluted or
supplemented with more organisms
Streaking and Placement of disk

1. Plate must be turned 60 degrees between 5. Within the 15minutes of disk placement,
each streaking ( overlapping) plates are inverted and incubated at 35C for 16-
2. The surface of the medium should be 18hours to maximum of 24hrs. Plates are
allowed to dry for 3 to 5 mins but not inverted to avoid the moisture contamination on
longer than 15 minutes the agar surface that can interfere with the
3. Within the 15 mins, the antimicrobial interpretation of test results
agents are applied
4. The disk must be positioned no closer
than 24mm from disk center to disk
center and no closer than 10-15mm from
the edge of the plate. The disk are pressed
firmly to ensure contact with the agar.
Measuring the zone of inhibition

1. The diameter of each zone of inhibition zone is measured using a caliper or

ruler
2. Zones are measured from the back side of the plate
3. For media that contains blood, the plate is examined with lid remove
6. Zone measurement is compared with interpretative tables of CLSI and
the results are interpreted as SUSCEPTIBLE, INTERMEDIATE , or
RESISTANT

a. Susceptible - the patient’s infecting organism should respond to therapy

with the antimicrobial agent using the recommended dosage for the site
of infection
b. Intermediate - microorganisms falls into a range of susceptibility in
which the MIC approaches or exceeds the antimicrobial agent level that
can be achieved
c. Resistant -no zone of inhibition or the inhibition did not meet the criteria
fro susceptible and intermediate
- Signifies that the antibiotics are not the appropriate choice for
treatment
CAUSES OF FALSE RESISTANCE

1. Use of heavy inocula or undiluted specimen

2. Late examination of plates ( prolonged incubation)
3. Use of disk with inadequate drug concentration ( Prolonged drug storage)
4. Failure to immediately refrigerate the disk after use
5. Use of wrong bacterial isolate
Factors affecting the zone of inhibition

1. Amount of inoculum or test organism

- Only pure culture can be tested
- Too light: false susceptibility too heavy : false resistant

1. Thickness of the agar plate ( 4mm)

Agar too thick : zone sizes will be smaller

Agar too thin ; zone will be larger

3. Growth rate of the test organism - rec. 35C with 16-18hrs of incubation

4. pH of the medium (7.2-7.4)

- High pH = decrease activity of tetracycline

- Low pH = decrease activity of aminoglycosides and erythromycin

5. Number of disk per plate - 150mm plate can have maximum of 12 disk

= greater than 12 may cause overlapping zones

6. Concentration of divalent cations ( Ca and Mg) in susceptibility media

High conc of Ca and Mg = dec activity of aminoglycosides against P. aeruginosa

= dec activity of tetracyclines against all bacteria