Assignment Microbiology Lab

ASSIGNMENT MICROBIOLOGY LAB
ASSIGNMENT TOPIC: CULTURE TRANSFER TECHNIQUES

PREPARED BY:
NAME: HAMMAD HUSSAIN SHAH
ENROLLMENT NO: 5-2/2021/098
SUBMITTED TO:
DR. MAQSOOD AHMAD KHAN
CULTURE TRANSFER TECHNIQUES:
INSTRUMENTS REQUIRED:
 Pipette, Inoculating needles or Loops, Bunsen burner, Petri dish,
Culture media tube.
CULTURE:
 To cultivate, or culture, microorganisms, one brings in a little sample
(the inoculum) into a container of nutrient medium, which provides a
suitable growth environment this process is called inoculation.
 The visible growth that later appears in or on the medium is known as
a culture.
 Note: prepare media and store in the refrigerator and use it in the time
of need and be careful to avoid contamination.
PRINCIPAL CULTURE TECHNIQUES:
1. Prepare the bench by disinfecting its surface with the available
disinfectant in the laboratory.
2. Label the bottom surface of a sterile Petri plate or the slant with your
name and date. Use china marking pen.
3. Flame the inoculating loop or needle over a Bunsen burner until the
wire becomes red-hot. Cool the hot loop in the broth culture until it
stops “hissing.”
4. Pass the sterile inoculating loop in a suspected sample. Inoculate the
Petri plate or slant by streaking as in next figure (3) by loop or
needle .or stabbing the agar deep tube.
5. If you used the test tube (slant), you should flame the neck of the tube
and then cap but not closed strictly?? if you used a Petri plate, you
should cover the plate directly after streaking
6. Incubate the slant or Petri plate in the incubator at 37 C for 24-84
hours. The Petri plates were incubated in an inverted position.
7. Note Incubation of the plate in an inverted position because Failure to
cool agar media before pouring into the plate will result in
condensation of moisture on the cover. Any moisture on the cover is
undesirable because if it drops down on the colonies, the organisms of
one colony can spread to other colonies, failing the isolation technique
CULTURING METHODS:
1. Streaking
2. Stabbing
3. Spread plate technique
SPREAD PLATE TECHNIQUE:

 The spread plate technique is the method of isolation and enumeration
of microorganisms in a mixed culture and distributing them evenly.
The technique makes it easier to quantify bacteria in a solution.
 A small volume of liquid, a diluted sample is pipetted onto the surface
of the medium and spread around evenly by a sterile spreading tool
(sometimes called a “hockey stick”)
USES OF SPREAD PLATE TECHNIQUE:

 It is used for viable plate counts, in which the total number of colony-
forming units on a single plate is enumerated.
 It is used to calculate the concentration of cells in the tube from which
the sample was plated.
 Spread plating is routinely used in enrichment, selection, and
screening experiments.
STABBING TECHNIQUE:
 Stab cultures are similar to agar plates but are formed by solid agar in
a test tube.
 Using a sterile straight wire, pick a single colony from a freshly
streaked plate or from a fresh culture, and stab it deep down into the
soft agar several times.
 Incubate the vial at 37°C for 8–12 h leaving the cap slightly loose.
 Seal the vial tightly and store it in the dark, preferably at 4°C.
USES OF STABBING:
 Stab cultures are most commonly used for short-term storage or
shipment of cultures.
STREAK PLATE METHOD:
 The streak plate technique is used for the isolation into a pure culture
of the organisms (mostly bacteria), from a mixed population. The
inoculum is streaked over the agar surface in such a way that it “thins
out” the bacteria. Some individual bacterial cells are separated and
well-spaced from each other.
 As the original sample is diluted by streaking it over successive
quadrants, the number of organisms decreases. Usually, by the third or
fourth quadrant, only a few organisms are transferred which will give
discrete colony forming units (CFUs).
PURPOSE OF STREAK PLATE METHOD:
 To produce isolated colonies of an organism (mostly bacteria) on an
agar plate. This is useful when we need to separate organisms in a
mixed culture (to purify/isolate a particular strain from contaminants)
or when we need to study the colony morphology of an organism.
 To identify the organism: biochemical tests to identify bacteria are
only valid when performed on pure cultures.
ISOLATION OF PURE CULTURES:
 When we try to culture samples, we soon discover that bacteria is
mixed and called a mixed culture.
 and we need to isolate one species isolation means that a single
bacterial cell is separated from other cells and provided enough space
on a nutrient surface, it will grow into a discrete colony return to
single known species or type of microorganism and called pure
culture.
 Subculture technique to make a second-level culture, a selected
colony may be picked up with an inoculating needle and transferred to
separate culture tubes.
PURE CULTURE METHODS:
1. STREAK PLATE METHOD: (describe above)
2. POUR PLATE METHOD:
 The original sample is diluted several times to reduce the
microbial population sufficiently to obtain separate colonies
upon plating. One advantage of this method is that it
requires somewhat less skill than that required for a good
streak plate; a disadvantage, it requires more media, tubes,
and plates.

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ASSIGNMENT MICROBIOLOGY LAB

ASSIGNMENT TOPIC: CULTURE TRANSFER TECHNIQUES

SPREAD PLATE TECHNIQUE:

USES OF SPREAD PLATE TECHNIQUE:

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