Transcript Environmental Microbiology -Laboratory Manual

Environmental Microbiology
-Laboratory ManualI
Isolation and Purification
Use of Microscope
Simple Stain
ENVIRONMENTAL MANAGEMENT TECHNOLOGY
FACULTY OF CIVIL AND ENVIRONMENTAL ENGINEERING
ITB, 2010
[email protected]
Isolation and Purification
Exp. 1 - Aseptic Culture Transfer
Exp. 2 – Pure Culture Isolation Technique
Exp. 3 – Isolation from Environment
Why Isolating ?
 Microorganism is everywhere
 Isolation : microorganism population  individual
species for study purpose  Pure Culture
Autoclave
Culture Tube
Laboratory Apparatus (1)
Petri Dish
Inoculation Needle
Bunsen Burner
Laboratory Apparatus (2)
Incubator
Broth
Medium
Semi-Solid
Solid
Deep Tube Agar
Solid Medium
Slant Agar
Plate Agar
Making Media
Aseptic Culture Transfer
Exp. 1 - Aseptic Culture Transfer
 Transferring m.o. culture from a medium to another
 standard procedure in microbiology lab
 M.o. are everywhere  external contamination 
ASEPTIC TRANSFER :
 Disinfection
 Bunsen Burner
Sterilizing Loop Using Bunsen burner
1.
) Position the handle end of the wire in the light blue cone of the flame
ii) Draw the rest of the wire upwards slowly into the hottest region of the flame –
immediately above the blue cone.
iii) Hold there until it is red hot.
iv) Ensure the full length of the wire receives adequate heating.
v) Allow to cool for a few seconds in the air, then use immediately.
vi) Do not put the loop down, or wave it around.
vii) Re-sterilize the loop immediately after use
Inoculation
Sterilizing Tube Mouth Using Bunsen Burner
Transfer from Liquid Medium to Broth Tube
In tube to tube transfers by loop or straight wire, both the tube containing the inoculums
source and the tube to be inoculated are usually in the hand at the same time.
Transfer from Slant Agar to Another
Transfer from Agar Media to Petri Dish
Pure Culture Isolation
 There is no individual species living by its own
 Laboratory  separated using pure culture to study
biochemical, morphology, etc
 Developed by Robert Koch
 Colony  m.o. mass growth seen on solid medium,
each colony represents multiplication of singe
organism  transferred to agar medium
 Streak plate, pour plate, spread plate
Streak Plate
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the dilution of bacteria by
systematically streaking them over the
agar surface in a petri dish to obtain
isolated cells
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During incubation, the isolated
microbes multiply
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Excessive moisture from the
condensation of water, derived from
the initial cooling of the hot sterile
media. If this water drops down onto
the agar surface, spreading and
merging of colonies can occur  one
should INVERT the plates after
streaking them and when they are
incubated.
Streaking Method
After Incubation
Pour plate
 also called the loop dilution method
 involves the successive transfer (serial dilution) of
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bacteria from the original culture to a series of tubes
of liquefied agar
the concentration of bacteria in the first tube is lower
than the concentration in the original culture
the process is repeated for a third tube of liquefied
agar
the contents of each tube is poured into a separate
Petri plate
a major concern in preparing pour plates is the
temperature of the agar
Spread Plate
Isolation from Environment
Isolation from Air
Isolation from Soil
Colony Counter
Using Microscopes
Terms & Definition
 A microscope  (from the Greek: mikrós, "small" and
skopeîn, "to look" or "see") = is an instrument to see
objects too small for the naked eye
 Microscopy  the science of investigating small
objects using such an instrument
 Microscopic  invisible to the eye unless aided by a
microscope.
 Optical, electron, etc.
Optical Microscope
 Using visible wavelengths of light
 the simplest and most used
 have refractive glass to focus light into the eye or
another light detector
 Typical magnification of a light microscope is up to
1500 x with a theoretical resolution limit of around 0.2
micrometers or 200 nanometers
 Caution 
 use of immersion oil in 100x magnification
 Coarse focus and fine focus
Components of A Microscope
A microscope consists of non optic components & optic components
Simple Stain
Staining
 Bacteria  extremely small m.o., transparant
 Staining is an auxiliary technique used in microscopy
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to enhance contrast in the microscopic image,
highlighting structures in biological tissues for viewing
Basic principle  ion bonding of cellular component
and active component of dye
Simple Stain & Differential Stain
Acid and base stain
Acid : dye (-), chinese ink, Nigrosin
Base : dye (+), fuchsin carbol, methylene blue
Preparing Smear
Heat fixation aims to attach m.o. to the slide, and to preserve the shape of the cells or tissue
involved as much as possible
After placing several drops of water on the slide, smear the bacteria from the inoculating loop
from the top to the bottom of the slide
Stain
Morphological Shapes of Bacteria
SAFETY FIRST !`
Work steril is the success of microbiology laboratory practice.