Appendix 3: Laboratory techniques

Different types of specimens collected for culture:

1. Urine: an early morning, mid stream urine sample collected in a sterile container, with precautions to prevent contamination. 2. Faeces: collected in a plastic container, or a rectal swab. 3. Sputum: a morning specimen in a sterile wide-mouthed container. If TB is suspected, collect specimens on 3 consecutive days. 4. CSF: collected by lumbar puncture, into a sterile container. 5. Serous fluids: collected in a sterile container, with citrate to prevent clotting. 6. Blood cultures: aseptically collected blood is injected using a syringe, into screw-capped bottles, with a rubber seal and a perforated metal cap.

Swabs: are used to collect samples from infected sites. The swab is inserted or rubbed over the infected site, including wounds (pus), ear, nasal passage, throat, eye, cervix, urethraetc.

Transport of specimens:
Specimens need to be sent to the laboratory without delay, since some organisms die off quickly outside the body and some may overgrow, giving a false result. If delay cannot be avoided, all specimens (except blood cultures and CSF, which should be kept at 37C) should be kept cool. Swabs should be placed in transport medium to preserve the organisms.

Aseptic handling of inoculating loop / straight wire

Wire loops and straight wires are used routinely for inoculating microbial cultures. It is made of nichrome, which has the advantage of heating up and cooling down rapidly and does not corrode. Before and during inoculation the wire is heated to kill any contaminating microbes.

Flame sterilizing the wire loop

Turn on the Bunsen burner. Hold the wire almost vertical and introduce the wire into the hottest part of the flame until it gets red hot (for about 15 seconds). Let the wire cool.

Sterilize the wire loop in hottest part of flame

Inoculation on agar plates for single colonies

Whole plate technique:
1. Label the plate and make sure the surface of agar is dry. 2. Flame sterilize the wire loop by inserting the loop in the cold region of flame and then slowly moving upwards to the hot region. Allow it to cool. 3. Place a small amount of inoculum near the periphery of plate, with a loop or swab. 4. Spread the inoculum over a small area (zone1), by a to- and- fro movement of the loop or swab. 5. Turn the plate to 45; overlap the previous streaks and streak on zone 2. 6. Flame the loop and allow it to cool before each streaking. 7. Repeat step-5, each time turning the plate to 45, over zone 3 and 4 8. Finally, streak the center of the plate with a ziz-zag movement in zone 5 9. Finally flame the wire loop. 10. Incubate the plates at the appropriate temperature and atmosphere, for the desired period of time.

Whole plate

3rd set of streaks

Primary inoculum Final set of streaks

1st set of streaks

2nd set of streaks

Half plate technique:

1. Using a marker pen, divide the plate into two halves. 2. Label the plate and make sure the surface of agar is dry. 3. Flame sterilize the wire loop by inserting the loop in the cold region of flame and then slowly moving upwards to the hot region. Allow it to cool. 4. Place a small amount of inoculum near the periphery of plate, with a loop or swab. 5. Spread the inoculum over a small area (zone1), by a to- and- fro movement of the loop or swab. 6. Turn the plate to 45; overlap the previous streaks and streak on zone 2. 7. Flame the loop and allow it to cool before each streaking. 8. Repeat step-5, each time turning the plate to 45, over zone 3. 9. Finally, streak the center of the plate with a ziz-zag movement in zone 4 10. Finally flame the wire loop.

Half plate
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Identification: Colony morphology

a) Shape: maybe circular, irregular, radiatingetc.

Circular

irregular

spindle

b) Surface: smooth, rough, fine granular, shiny dulletc.

c) Edge or margin: the edges of the colonies can be described as smooth, entire, filamentous, rough or rhizoid, or irregularetc.

Entire/smooth

undulate

lobate

filamentous

d) Elevation: this can be determined by tilting the plate and looking at the side of the colonies. The colonies maybe raised convex, flat, umblicatedetc.
Raised Flat Convex Umbonate

e) Size: large, medium, small, pinpoint etc. usually the colonies are 2-3 mm in diameter, smaller ones maybe less than 1mm.

f) Optical characteristics: the colonies may be: Transparent, translucent, opaque, iridescent, dull, glossy

g) Consistency: this is determined by touching the colonies with a sterile loop. The colonies may be mucoid, tenacious, dry, brittle, creamy or waxyetc. some may be adherent to the loop

h) Color and Pigmentation: used to describe a particular genus in general. Colonies may be white, gray, yellow, or buff. Some organisms produce pigmented colonies. When pigments diffuse into agar around the colony its known as a soluble pigment. Insoluble pigments are those that remain in the colony.

i) Odor: some organisms produce distinctive odors.

j) Haemolysis: a reaction observed on blood agar plate, seen as a clearing of blood around the colonies. Three types of hemolysis are shown by bacterial species. Alpha: greenish zone of partial hemolysis around colonies. Beta: clear zone of complete hemolysis around colonies. Gamma: no hemolysis.

k) Contiguity: colonies maybe discrete or swarmingetc.

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