laboratory manual - .2 1. instructions to the microbiology laboratory and good laboratory practices
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DEPARTMENT OF MICROBIOLOGY
ST ALOYSIUS COLLEGE (AUTONOMOUS), MANGALURU
INSTRUCTIONS TO THE MICROBIOLOGY LABORATORY AND GOOD
LABORATORY PRACTICES. 2
2 STUDY OF GLASSWARE USED IN MICROBIOLOGICAL WORK 4
STUDY OF MICROBIOLOGY LABORATORY EQUIPMENT
4 MICROSCOPY 17
5 PREPARATION OF BACTERIAL SMEARS 24
6 SIMPLE STAINING 27
7 GRAM STAIN 29
CAPSULE STAINING WET INDIA INK METHOD AND DRY INDIA INK
9 ENDOSPORE STAINING METHOD- SCHAEFFER FULTON METHOD 36
DEMONSTRATION OF BACTERIAL MOTILITY BY HANGING DROP
PREPARATION OF CULTURE MEDIA: NUTRIENT BROTH AND
NUTRIENT AGAR 41
SERIAL DILUTION TECHNIQUE. VIABLE COUNT BY STANDARD PLATE
COUNT METHOD 46
13 ISOLATION BY STREAK PLATE METHOD 49
14 STUDY OF COLONY MORPHOLOGY (CULTURAL CHARACTERISTICS). 50
1. INSTRUCTIONS TO THE MICROBIOLOGY LABORATORY AND GOOD LABORATORY PRACTICES.
1. Wash your hands with disinfectant soap when you arrive at the lab and again before you leave.
2. Absolutely no food, drinks, chewing gum, or smoking is allowed in the laboratory.
3.Do not put anything in your mouth such as pencils, pens, labels, or fingers.
4.Do not store food in areas where microorganisms are stored.
5. Purchase a lab coat and safety glasses, bring them to class, and use them. Alternatively, a long sleeved
shirt that buttons or snaps closed is acceptable protective clothing.
6. Avoid loose fitting items of clothing. Wear appropriate shoes (sandals are not allowed) in the
7. Keep your workspace free of all unnecessary materials.
8. Disinfect work areas before and after use with 70% ethanol or fresh 10% bleach. Laboratory equipment
and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, and
especially after spills, splashes, or other contamination.
9. Label everything clearly. 8. Replace caps on reagents, solution bottles, and bacterial cultures.
10. Do not open Petri dishes in the lab unless absolutely necessary.
11. Inoculating loops and needles should be flame sterilized in a Bunsen burner before you lay them down.
12.. Turn off Bunsen burners when not in use.
13.Long hair must be restrained if Bunsen burners are in use.
14. Treat all microorganisms as potential pathogens.
Use appropriate care and do not take cultures out of the laboratory.
15. Wear disposable gloves when working with potentially infectious microbes or samples (e.g., sewage).
If you are working with a sample that may contain a pathogen, then be extremely careful to use good
16. Sterilize equipment and materials.
17. Never pipette by mouth. Use a pipetting aid or adjustable volume pipettors.
18. Do not lick labels. Use only self-stick labels for the identification of experimental cultures.
19. Consider everything a biohazard. Do not pour anything down the sink. Autoclave liquids and broth
cultures to sterilize them before discarding.
20. Dispose of all solid waste material in a biohazard bag and autoclave it before discarding in the regular
21.. Familiarize yourself with the location of safety equipment in the .
22. Dispose of broken glass in the broken glass container.
23.Dispose of razor blades, syringe needles, and sharp metal objects in the sharps container.
24. Report spills and accidents immediately to your instructor. Clean small spills with care .
25. Report all injuries or accidents immediately to the instructor, no matter how small they seem.
2. STUDY OF GLASSWARE USED IN MICROBIOLOGICAL WORK
1)CULTURE TUBES AND PETRI DISHES
Glass test tubes and glass or plastic Petri dishes are used to
cultivate microorganisms. A suitable nutrient medium in the form
of broth or agar may be added to the tubes, while only a solid
medium is used in Petri dishes. A sterile environment is
maintained in culture tubes by various types of closures.
Historically, the first type, a cotton plug, was developed by
Schreder and von Dusch in the nineteenth century. Today most
laboratories use sleevelike caps (Morton closures) made of metal,
such as stainless steel, or heat-resistant plastics. The advantage of
these closures over the cotton plug is that they are labor-saving
and, most of all, slip on and off the test tubes easily.
2) Petri dishes provide a larger surface area for growth and
cultivation. They consist of a bottom dish portion that contains the medium and a larger top portion that
serves as a loose cover.
For routine purposes, dishes approximately 15 cm in diameter are used.
The sterile agar medium is dispensed to previously sterilized dishes from
molten agar deep tubes containing 15 to 20 ml of medium, or from a
molten sterile medium prepared in bulk and contained in 250-, 500-, and
1000-ml flasks, depending on the volume of medium required. When
cooled to 40C, the medium will solidify. Remember that after inoculation, Petri dishes are incubated in an
inverted position (top down) to prevent condensation formed on the cover during solidification from
dropping down onto the surface of the hardened agar.
3) Pipettes: A pipette is another instrument used for aseptic transfers. Pipettes are similar in function to
straws; that is, they draw up liquids. They are made of glass or plastic drawn out to a tip at one end and
with a mouthpiece forming the other end. They are calibrated to deliver different volumes depending on
requirements. Pipettes may be sterilized in bulk inside canisters, or they may be wrapped individually in
brown paper and sterilized in an autoclave or dry-heat oven.
4). Slides: A microscope slide is a thin flat piece of glass, typically 75 by 26 mm (3 by 1 inches) and about
1 mm thick, used to hold objects for examination under a microscope.
Cavity Slide: These have a depression in the slide. These slides are useful as moist
chambers for observing hanging drops.
5) Cover slips: These are a thin flat piece of transparent material,usually square or rectangular about 20
mm in wide a nd fraction of mm thick that is placed over objects for viewing with microscope.
6) Measuring cylinders,7) Conical Flask,8) Volumetric Flask, 9) Beaker,10) Durham tubes .11)Bent
glass rod( Spreader), 12) Glass rod.
Transfer Instruments: Microorganisms must be transferred from one vessel to
another or from stock cultures to various media for maintenance and study. Such a
transfer is called subculturing and must be carried out under aseptic conditions to
prevent possible contamination.
Wire loops and needles are made from inert metals such as Nichrome or platinum
and are inserted into metal shafts that serve as handles. They are extremely durable
instruments and are easily sterilized by incineration in the blue(hottest) portion of the Bunsen burner
3. STUDY OF MICROBIOLOGY LABORATORY EQUIPMENT /INSTRUMENTS
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 glasswares, 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 100C
(boiling point of water). 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 100C 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
In operating a standard vertical autoclave, sufficient water is poured into it. If water is too less, the bottom
of the autoclave gets dried during heating and further heating damages it. The materials to be sterilized
are covered with craft paper and arranged on an aluminium or wooden frame kept on the bottom of the
autoclave, otherwise if the materials remain half-submerged or floating, they tumble during boiling and
water may enter. The autoclave is closed perfectly airtight only keeping the steam release valve open.
Then, it is heated over flame or by the in-built heating element. Air inside the autoclave should be allowed
to escape completely through this valve. When water vapour is seen to escape through the valve, it is
Temperature and pressure