SSCT 1611 Microbiology Lab

Data Worksheet

Name : Section :
Student ID : Group number :

Instruction
1. The worksheet shall be completed as individual work.
2. The cumulative carry marks from all worksheet for the whole course is 25%.
3. Every worksheet must be handed in on designated lab session for markings.

Due on
Dec 5th, 2023 10 a.m.

Experiment 3 Isolation of Pure Culture (making broth culture, streak plates, and
slant cultures)
Experiment 4 Morphological Observation of Microbial Cells (bacterial cell
staining and microscopy techniques)

Learning Objectives
1. Demonstrate correct and accurate technique of streaking and spreading;
2. Observe and record colony morphology of pure bacterial colonies grown on solid medium;
3. Differentiate the morphology and ability of bacterial strains to grow on enrichment and
minimal media.
SSCT 1611 Microbiology Lab
Data Worksheet

Table 3.1 Morphology of isolated microbes

Plate/ liquid/ slant culture Colony morphology
Example:
Yellow circular colony with convex
elevation, entire edge and smooth surface.

Example:
Filamentous colony with raised elevation
and fuzzy edge. White at the centre and
translucent white at the edge.

Example:
Creamy or off-white circular colony with
irregular elevation, curled edge and
concentric surface.
SSCT 1611 Microbiology Lab
Data Worksheet

Plate/ liquid/ slant culture Colony morphology

SSCT 1611 Microbiology Lab
Data Worksheet

Table 4.1 Morphology of isolated bacteria and Table 4.2 Group of bacteria based on Gram-staining
From Experiment 3 Experiment 4
Plate/ liquid/ slant culture Colony morphology Cell morphology
Example A (From Exp Example
3)
Dark blue/purple cells
Yellow circular (Gram positives), coccus
colony with convex in tetrad. Magnification
elevation, entire edge, 1000x
and smooth surface.

OR Example

Example B (From Exp Dark blue/purple cells

2) (Gram positives) coccus in
tetrad. Magnification
(arrow) Yellow 1000x
circular colony with
convex elevation,
entire edge, and
smooth surface.
SSCT 1611 Microbiology Lab
Data Worksheet

From Experiment 3 Experiment 4

Plate/ liquid/ slant culture Colony morphology Cell morphology
SSCT 1611 Microbiology Lab
Data Worksheet

Table 4.3 Spore formation of bacterial culture

Culture Spore formation (Yes/No)
Broth Example:

Yes, there were spores.

Bacilli cells (blue arrow) and spores (black
arrow). Magnification 1000x
SSCT 1611 Microbiology Lab
Data Worksheet

Post experiment reflections

1. Figure 1 is an expected streak plate. Compare your own streak plate with Figure 1.

Figure 1
a. Was your streak plate a success? Explain your answer. [4 marks]
No,this is because there is no presence of pure culture colony and there is no distinct or well-
defined spot can be found on the agar plate.There is also some discoloration which shows a
sign of contamination.

b. Does your streak plate contain pure culture? Explain your answer. [4 marks]
No,my streak plate does not contain pure culture but contain mixed cultures because there is
no distinct and obvious spot can be found on the plate showing a colony.The spots on the plate
are very close to each other so,I cannot identify where and which is the single colony.

2. Did you get any spores from your broth culture? If yes, explain if it is endospore or spores?
If no, give one reason why you did not observe any spores?
No, the reason might be the bacterial species in the culture may not be capable of forming
spores or the culture conditions may not have been conducive to spore formation.

3. Compare and contrast the chemical reactions behind the different staining protocols that
you used to stain bacterial cells. [6 marks]
SSCT 1611 Microbiology Lab
Data Worksheet

The staining protocols used to stain bacterial cells are Gram staining and staining for bacterial
endospores.Gram staining is a differential staining technique that separates bacteria into two
groups based on the structure of their cell walls. Malachite green is commonly used in the
endospore staining technique, which is a differential staining technique used to identify
bacterial endospores.Gram-positive bacteria have a thick peptidoglycan layer in their cell
walls,which retains the crystal violet stain and appears purple under the microscope,while
Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane that does not
retain the crystal violet stain,but instead it takes up the counterstain safranin and appears pink
or red under the microscope. The endospore staining technique involves the use of heat to drive
the malachite green stain into the endospores, followed by a counterstain with safranin to stain
the vegetative cells.The chemical reactions of Gram staining involve the use of crystal
violet,safranin,iodine and alcohol while behind the endospore staining technique involve the
use of malachite green, heat, and safranin.In contrast, Gram staining is a differential staining
technique that separates bacteria into two groups based on the structure of their cell walls, while
endospore staining is used to identify bacterial endospores.