MIC60104 Tutorial 1 August Semester, 2017

Introduction to microbiology

1. Construct a table to show 3 major differences between the cell wall of gram positive and negative bacteria.
[6 marks]
Wall Materials Gram +ve Gram -ve
Peptidoglycan Thick Monolayer/Thin
Lipopolysaccharide No Yes
Outer Membrane No Yes

2. Explain how gram staining procedure is used to differentiate 2 groups of bacteria based on gram reactions.
[6 marks]
-Thick peptidoglycan retains crystal violet-Iodine complex after alcohol washing
-Cells are stained purple-blue as Gram +ve
-Thin peptidoglycan are porous and not bale to retain CV-I complex after decolourization
-Cells are stained pink with counterstain as Gram -ve

3. Draw the following shape and arrangement of bacterial cells. [10 marks]
I. Coccus
II. Streptococcus
III. Staphylococcus
IV. Bacillus
V. pleormophic-mycoplasma

4. You are assigned to detect the presence of the following cell structures of an unknown microorganism
isolated from a water faucet at Taylor's University Lakeside Campus.
Explain how do you work out the test.
[6 marks]
I. Peptidoglycan
-Prepare a fixed bacteria smear and perform the following sequential steps:
Crystal violet (30 s) iodine mordant (1 min), alcohol wash, safranin (30 s)
-Purple-blue: Gram +ve; Pink: Gram -ve

II. Capsule
-Prepare a fixed bacteria smear and perform the following sequential steps:
India ink to negatively stain the background and counterstain the cells with safranin
-Capsule appears as hollow zone surrounding the pink cells

5. You received a 10-ml sample of dairy product which is suspected to be the cause of food poisoning
incidence in a primary school at Johor Bahru. Describe the procedure for the following tests;

I. Estimation of the total viable count of microorganism in dairy sample [3 marks]

-Sample will be diluted, serially, at 10-fold
-1 ml of each diluted sample will be plated out by pour plate method in molten nutrient agar
and incubated ON
-Plates with 30-300 colonies will be scored by visual inspection and bacterial count will be
MIC60104 Tutorial 1 August Semester, 2017

calculated by multiplying the averaged score with respective dilution factor.

II. Presumptive identification of microorganism in the sample, assuming the broth that you are using
supports growth of all microorganisms [3 marks]
-Enrich the growth of microorganisms in broth
-Examine the cultures with simple, differential and specific staining to identify the shapes,
cellular arrangement and gram reaction.

III. Isolate pure culture of microorganism, assuming that the dairy sample contains mixed culture of
microorganisms [3 marks]
-Streak out the enriched culture from (ii) on appropriate agar to isolate single colonies