PRACTICAL 3: STERILIZATION
Objective
At the end of the practical, the students should know the following;
How to sterilize using
Physical method
Chemical method
TYPE METHOD USED MATERIAL
PHYSICAL Dry heat Hot air oven Glass wares e.g.
Flaming Petri dishes
Pipettes, flasks, wire
Loops, spreaders,
forceps
0
Moist heat Below 1000 c Heat labile material
0
Steaming at 1000 c Non autoclavable
Above 10000c Culture media
autoclaving
Filtration Membrane filtres
CHEMICAL Gas Formaldehyde Equipment e.g.
safety cabinet
Liquid Hypochlorite Surfaces, hands
solution
STERILIZATION BY AUTOCLAVING
Method
Add correct volume of water to the autoclave.
Place the wire bottles containing the bottles with loose caps in the inner chamber of
the autoclave.
Secure the lid, open the air lock and close the draw off knob
Adjust the safety valve to the required pressure
Switch on the equipment, as the water boils air and steam emerges through the air
lock.
When all water droplets have been expelled and only steam is emerging, wait for 1
min and close the air lock.
When the required pressure has been reached and the excess steam begins to be
released from the safety valve, reduce the heat and start timing.
At the end of the timing, turn off the heat and allow the autoclave to cool until the
pressure gauge is showing zero.
Open the air lock and wait for a few minutes before opening the lid.
Remove the lid and tighten the bottle caps.
Question
Briefly discuss various methods used as control of an autoclave
PRACTICAL 4
MEDIA PREPARATION
You are provided with the following culture media
LIQUID
SOLID
SEMI-SOLID
MacConkey agar
Blood agar base for CBA and BA
STA (Muella hinton agar)
TSI
REQUIREMENT
Carry out the following steps as indicated on the culture media container.
i. Weighing and dissolving
ii. Addition of heat sensitive ingredients
iii. Dispensing
iv. Sterilization and sterility testing
v. Storage
OBJECTIVES
At the end of the practical the student should be able to;
1) Explain the purpose of the ingredients in various media.
2) Explain the use of various culture media.
3) Describe the various methods of preparing culture media.
4) Explain the various storage methods of prepared culture media.
PRACTICAL 5
INOCULATION AND INCUBATION OF CULTURE MEDIA
Objectives
At the end of the practical, the student should be able to;
1. Carry out the various culture methods.
2. Explain the factors influencing the growth of micro-organisms
Aseptic technique,
Flame sterilizes wire loops, straight wires and metal forceps before and after
use.
Flame the necks of the specimen bottles, culture bottles and tube after
removing and before replacing caps.
Caps of bottles should not touch unsterilized surface.
Make slide preparation from specimen after inoculating the culture media.
Decontaminate the work surface before and after use.
Wear protective clothing, wash hands after handling infectious material.
DAY 1
Inoculation of media in Petri dishes
Apply the inoculum to a small area of a plate.
Flames sterilize the loop and let it cool.
Spread and thin out the inoculum as indicated in the diagram
Inoculation of slopes and butt
Use a sterile straight wire to stab into the butt first and then use the same wire to streak the
slope in a zig zag pattern
Inoculation of fluid media
Use a sterile straight wire, wire loop or Pasteur pipette to inoculate fluid media.
Incubate all cultures at 370C for 24 hours
DAY 2
Examine the culture plates, tubes and bottles for:
Pigmentation
Consistency
Margin
Elevation
Size and note down results
Prepare smear on the slide from the plate and perform the following staining techniques:
Gram staining technique
Hiss staining technique
Determine whether the bacteria are motile or non-motile by performing motility test
using the fluid culture media.
