MICROBIOLOGY

Lesson 02
BASIC LABORATOTY
TECHNIQUES

 Sterilization techniques
 Preparation of artificial culture media
 Bacteria staining

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 METHODS OF STERILIZATION
What is sterilization?

The process of removal or destruction of all forms of microbial life including endospores

Two types: 1) Physical methods

2) Chemical methods

Physical methods of sterilization

• Moist heat sterilization

• Dry heat sterilization
• Pasteurization
• Boiling
• Membrane filtration
• UV radiation
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 1. Moist heat sterilization
Moist heat is used to destroy microorganisms present in materials like culture media,
temperature stable reagents/ fluids and laboratory utensils.

Principle: Microorganisms are destroyed by denaturing

of proteins by high temperature and pressure

Autoclaving:

Conditions applied:
Steam with 121 °C temperature
At the pressure of 1 atm/ 15 psi
for 15 minutes in an autoclave

This kills all microorganisms

and their endospores (except prions)
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Materials sterilized using autoclaving:
(Items that withstand high temperature
and pressure)

• Culture media
• Water
• Solutions
• Syringes and needles
• Health care instruments
• Glassware
(Ensure that the steam contacts
all surfaces

Pressure cooker also can use for

moist heat sterilization
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2. Dry heat sterilization
Used to destroy microorganisms present in materials like glassware, petri dishes,
pipettes, inoculation loops, inoculation needles, scalpels.

Three methods of dry heat sterilization;

(a) Direct flaming
Materials sterilized using direct flaming:

Inoculation loops
Inoculation needles
Scalpel blades

Materials are heated on flames of Bunsen burner / hot spirit lamp until they reach red hot

Principle: Microorganisms are burned into ash

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 (b) Incineration
• Done in an incineration oven
• Used to sterilize hospital waste
• Principle: Microorganisms are burned into ash

(c) Hot-air sterilization

Conditions applied:
Heated to about 170 °C for 2 hours
In a dry air oven
• Principle: Microorganisms are killed by
oxidation
Materials sterilized using hot-air sterilization
Glassware like Petri dishes, flasks, beakers, Hot air oven
Bottles, and glass pipettes
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3. Pasteurization
Objective: To eliminate pathogenic microorganisms and reduce microbial number which
prolongs milk quality under refrigeration

Ultra-high temperature (UHT)

140 °C in less than 5 seconds

High temperature short time (HTST)

72 °C for 15 seconds

Low temperature long time (LTLT)

63 °C for 30 minutes
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 4. Boiling
• Materials like surgical instruments are boiled to 100 °C

• Most of pathogenic microorganisms are killed at boiling

temperature
5. Filtration – Membrane filters
Membrane filters are widely used to sterilize heat sensitive solutions
Ex: Solutions containing enzymes, vitamins, antibiotics, vaccines
Some culture media (Heat labile culture media)

Principle: Solution is passed through the filter using

vacuum
Pores of membrane filters are from 0.01 µm to 0.45 µm
size
Filter retain almost all microorganisms including viruses
and some large protein molecules THARAKA MUTHUNAYAKE BIOLOGY
6. UV radiation
Principle: Kills microorganisms that falls into direct exposure, either through destruction or
damaging DNA

Disadvantage: UV radiation doesn’t penetrate solid surfaces and covering like paper, glass
and textile. Therefore, anything to be sterilized should have direct contact with radiation

Used to sterilize air in hospital rooms like operating theaters and nurseries

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 Chemical methods of sterilization
Currently using chemical sterilizing agents: Ethylene oxide & Chlorine dioxide
Principle: Reduce microbial populations to safe levels or remove vegetative forms of pathogens

• Ethylene oxide kills microorganisms and • Chlorine dioxide is used to fumigate

endospores enclosed building areas contaminated
• Highly penetrating with endospores of Bacillus anthracis
• Used to sterilize mattresses in hospitals • Commonly used in water treatment prior
to chlorination
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 PREPARATION OF CULTURE MEDIA
To study microorganisms they should be brought to laboratory and should provide similar
conditions for their growth and reproduction.

Culture medium? A nutrient material prepared for providing nutrition and anchorage
essential to the growth of microorganisms at laboratory conditions.

A culture medium should contain; All microorganisms can’t be grown on

 Necessary nutrients laboratory culture media, which are called
 Sufficient moisture non culturable microorganisms
 Suitable pH
 Medium should initially sterile Some microorganisms grow well in culture
media whereas others require special
When preparing culture medium, all medium
glassware and liquid nutrient solutions
should be sterilized
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 Two commonly used culture media to grow bacteria and fungi

NUTRIENT AGAR POTATO DEXTROSE

AGAR
Used to grow bacteria Used to grow fungi

Components of culture medium: Components of culture medium:

• Peptone – 10 g • Potato – 200 g

Nutrients Nutrients
• Meat extracts – 10 g • Glucose – 20 g
• NaCl – 5 g • Agar -15 g – solidifying agent and provide
• Agar – 15g – solidifying agent and provide substrate for the growth of microorganisms
substrate for the growth of microorganisms • Distilled water – 1000ml
• Distilled water – 1000 ml pH – 5.6
pH – 7.2
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Practical 1: Preparation of microbial culture medium in laboratory
and culturing microorganisms

1. Sterilize 2. Prepare 3. Sterilize the solutions by

glassware using Nutrient agar autoclaving at 121 °C for
hot-air sterilization and PDA 15 min (15 lb/sqin.)
culture media

4. Pour about 15 ml of the

sterilized Nutrient Agar and
PDA into sterilized Petri dishes, 6. Label the bottom
using aseptic techniques 5. Set aside to
of each agar plate
solidify
using a marker pen

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 7. Flame the 8. Aseptically obtain a 9. Place a loop full of
inoculating loop loop full of the sample on the agar plate
to redness, sample. near the edge of the
allow it to cool Eg: toddy and yoghurt dish and streak
on the agar surface in a
zig zag pattern

10. Incubate for 24-48 hr.

at room temperature

OR

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 STAINING MICROORGANISM
Why staining is essential to observe microorganisms in light microscope?
Microorganisms appear almost colourless. When observed through light microscope they
do not produce contracting images.

• A simple stain is an aqueous or alcohol solution of a single basic dye.

 The primary purpose of simple staining:

To highlight the entire microorganism so that cellular shapes, cell arrangements and basic
structures are visible

 Some simple stains commonly use in laboratory:

Methylene blue, Crystal violet, Safranin

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Practical 2: Staining bacteria using simple staining (Methylene blue)

Main steps Purpose of the step

1. Preparation of thin smear on a clean glass slide - To Spread bacteria on the slide

2. Air drying the smear - To fix bacteria on to the slide

3. Heat fix the smear by passing the slide through - Enhance the adherence and to prevent
a flame two or three times remove out bacteria during washing

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4. Flood the prepared, heat – fixed smear with - To stain bacteria
2 or 3 drops of Methylene Blue
and allow 30-60 seconds

5. Wash with tap water - To remove the excess stain

Staining the smear with Methylene blue Microscopic examination

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