This document provides guidelines for practicing safe and aseptic techniques in a microbiology laboratory. It discusses wearing protective clothing and equipment like gloves and goggles. Maintaining cleanliness and orderliness in the lab is emphasized to avoid contamination and accidents. Common equipment used includes autoclaves for sterilization, incubators for culture growth, and laminar flow hoods. Proper labeling and disposal of materials is also covered.

1. Introduction To Practical
Microbiology

2. Safe Patterns
• Safe patterns include:
• Standard Lab Rules
• A septic Techniques

3. Protecting yourself
• Wear the clothing and protective wear identified in your risk
assessment
• Laboratory coats must be kept fastened
• Don’t wear sandals or open shoes

4. Protecting yourself - gloves
Remove your gloves before using instruments, telephone,
and leaving the laboratory

5. Laboratory hygiene
• Never eat, drink or smoke in a laboratory
• Never apply cosmetics
• Never touch your face, mouth or eyes
• Never suck pens or chew pencils
• Always wash your hands before you leave and especially before eating

6. Note
 When preparing or working with strong solutions from stock and
dealing with powder form, wear eye protection and gloves to avoid
irritant or harmful effects.

7. General Tidiness
X
 Clear up waste, deal with washing up and
put things away as you finish with them
 Make sure everything is safe before you
leave .
 A tidy laboratory avoids accidents to
everyone.

8. STERILIZATION vs DISINFECTION
Disinfection
 Sterilization means the complete destruction of all the microorganisms including
spores, from an object or environment.
 It is usually achieved by heat or filtration but chemicals or radiation can be used.
 Disinfection is the destruction, inhibition or removal of microbes that may cause
disease or other problems
e.g. spoilage.
 It is usually achieved by the use of chemicals.

9. • It is important in microbiology to work with pure cultures
• This is difficult because the world around us is covered with Micro organisms
(even on dust particles in the air)
• In order to protect broth, plates, slants and pure cultures from contamination
around us, we practice aseptic techniques

10. When culturing bacteria or other microorganisms, it is important to keep
your work area as clean as possible.
This prevents the introduction of other microorganisms from the
environment into your culture.

11. Aseptic Techniques

12. Aseptic technique
• “A” = Negative prefix
• “Septic” = Infection
• Aseptic technique refers to a procedure that is performed under sterile
conditions techniques to prevent bacterial contamination.

13. 1. Start by washing down your work or lab benches with a
surface disinfectant. The most commonly used
disinfectants for lab use are 50-80%% ethanol.

14. 2. Turn off any forced air heating or air conditioning units that create
strong air current in your work area.
3. A small room that can be closed off is worth the effort to set-up if
you will be doing a lot of microbial culturing.
4. You can install a UV bulb in a fluorescent light fixture to surface
sterilize your work bench if you have an enclosed area. Remember
to leave the area when you turn on the UV light source!

15. 5. All glassware should be cleaned and sterilized before you
begin.
6. All pipettes, spatulas, and test tube (culture) racks should
also be sterilized.
7. You can purchase sterile, disposable culture tubes, petri
dishes, and pipettes to minimize the quantity of glassware
that you have to sterilize.

16. • You should have only the materials you'll need .

17. Equipments:

19. Autoclave oven

20. Water bath
• Suitable temperature for keeping melted agar media molten for use
( 50 °C);
• accurate temperature control.

21. Microscope
• Microscope, slides, cover slips,
• stains, staining rack, immersion
oil.

22. Bunsen burner

23. Fridge (Refrigerator)
• Storage of heat-labile materials

24. Laminar hood

25. Sterilization of equipment and materials
Technique depends on article:
Does it burn or melt?
Do you want it back

26. • Wire loop: Heat the entire piece of metal of the inoculation
instrument to redness in Bunsen burner flame when using metal
inoculating loops. Be sure to COOL your inoculation instrument
before picking the inoculum (broth or agar).

27. Hot Air Oven:

28. • It is used for sterilization of glassware’s, such as test tubes,
pipettes and petri dishes (not plastic!) . Such dry sterilization is
done only for glassware’s. Liquid substances, such as prepared
media and saline solutions cannot be sterilized in oven, as they
lose water due to evaporation.

29. • The glassware’s are sterilized at 180°C for 1 hour. An oven has a
thermostat-control.
• 160ºC for 2 hours.
• it must be wrapped in aluminum and held to additional time to come
to temperature (and cool down!).

30. AUTOCLAVE
• The principle of sterilization in
an autoclave is that steam under
pressure is used to produce a
temperature of 121ºC which if
held for 15 minutes all
microorganisms including
bacterial endospores will be
destroyed.

31. • Autoclave is the nucleus of a microbiology laboratory. It is used
not only to sterilize liquid substances such as prepared media
and saline (diluents) solutions, but also to sterilize glassware’s,
when required.
• It has the same working principle as a domestic pressure
cooker. The maximum temperature that can be obtained by
boiling water in an open container is 100°C (boiling point of
water).

32. • This temperature is sufficient to kill only the non-spore formers, but it
is difficult to kill the spore-forming bacteria at this temperature, as
they escape by forming heat resistant spores. It takes very long time to
kill the spores at this temperature.
• On the other hand, when water is boiled in a closed container,
due to increased pressure inside it, the boiling point elevates
and steam temperature much beyond 100°C can be obtained.
This high temperature is required to kill all the bacteria
including the heat resistant spore-formers. Steam temperature
increases with increase in steam pressure

33. Microbiological Incubator:
• Profuse growth of microbes is obtained in the laboratory by growing
them at suitable temperatures. This is done by inoculating the desired
microbe into a suitable culture medium and then incubating it at the
temperature optimum for its growth.

34. • Incubation is done in an incubator which maintains a constant
temperature specifically suitable for the growth of a specific microbe.
As most of the microbes pathogenic to man grow profusely at body
temperature of normal human being (i.e. 37°C), the usual temperature
of incubation is 37°C.

35. • The incubator has a thermostat, which maintains a
constant temperature, set according to requirement.
Accurate temperature can be seen on the thermometer
fixed on the incubator.

36. Tips
• All agar plates are
incubated UPSIDE DOWN
to reduce bacterial
contamination and to
reduce the possibility of water
condensation that may be on the lid
dropping onto the agar, causing fluid to
run across the agar medium.

37. Tips
• Keep petri dishes and test tubes covered as much as possible.
• If top must be removed completely do not LAY IT on the lab top. This
lowers the probability of contamination and prevents “false positive”
results.

38. Tips
• Label all test tubes and petri plates with your name (initials), date,
and name of organism BEFORE you add any solutions, bacteria, etc.

39. Tips
• Do not dump ANY microbial suspension down the drain or in the trash
can. collect them for proper disposal.

40. Tips
• Place test tubes in racks
when working at your table:
never lay the tubes down—they leak.
• Keep test tube caps and petri dish covers to reduce contamination
(matters not whether it is sterile media or already cultured).

41. Tips
• Sterilize after preparation, in storage, or working containers. Handle
aseptically.