Universiti Teknologi MARA Cawangan Perak

Kampus Tapah

Faculty of Applied Sciences

Diploma in Science

BIO301

Practical 1:
Techniques in Microbiology

Lecturer:
DR Low Kim Fatt

Group:
A4AS1205_A

Student name:
Nurul Farhana binti Amir Fadzil

Student ID:
2017266116
Practical 1.1: Bacteria isolation using streaking method (Note: Max: 3 pages, Font: Times, Font size: 12)

Introduction

A culture medium is a liquid or gelatinous substance that contains essential nutrients,

to cultivate target microorganisms or tissues, for further purposes. A culture medium
must be sterilised before use so that no unwanted microorganisms grow, which may
contaminate the growing sample.
Streaking is a technique used to isolate a pure strain from a single species of microorganism,
often bacteria. Samples can then be taken from the resulting colonies and a microbiological
culture can be grown on a new plate so that the organism can be identified, studied or tested.

Methodology

1. The bench was once cleaned with 10% Milton and twice with 70% ethanol
2. The bunsen burner was switch on to establish the cone of sterility
3. Disinfected hands with 70% ethanol
4. The loop was sterilized
5. The loop was cool down by stabbing it in a clean part of the agar
6. A single colony was pick up
7. The colony was spread using the streak plate technique
8. The loop was sterilized and rest on a stand
9. The bottom of the plate was labelled with the isolate name and date of spreading
10. The plate was sealed with a double layer of parafilm
11. The plate was incubated upside-down at the desired temperature
12. The present of single colonies and absence of contamination was checked
Expected results

The streaked plate is incubated upside-down at desire temperature. At the end of incubation
there should be enough bacteria to form visible colonies in the area touched by the
inoculation loop. The colonies grown in the plate carefully. There are yellowish in colour at
the plate. All colonies have the same general appearance. It can be identified based on their
morphological differences.
Practical 1.2: Gram staining (Note: Max: 3 pages, Font: Times, Font size: 12)

Introduction

The Gram stain is a very important preliminary step in the initial characterization and
classification of bacteria. It is also a key procedure in the identification of bacteria based on
staining characteristics, enabling the bacteria to be examined using a light microscope. The
bacteria present in an unstained smear are invisible when viewed using a light microscope.
Once stained, the morphology and arrangement of the bacteria may be observed as well.
Furthermore, it is also an important step in the screening of infectious agents in clinical
specimens such as direct smears from a patient.
The Gram stain procedure enables bacteria to retain color of the stains, based on the
differences in the chemical and physical properties of the cell wall.

Methodology

1. Smears from cultures of at two type microorganisms were prepared

2. The slides on a staining for each smear were placed with crystal violet for 1 minute
3. The crystal violet from each slide was wash with tap water
4. Each smear was flooded with gram’s iodine for 1 minute
5. The gram’s iodine from each slide was washed with tap water
6. Each slide was decolorized with acetone alcohol until the slide appears colourless for
5 to 15 seconds
7. The slide was briefly washed with tap water
8. The smear was counterstained with safranin for about 1 minute
9. The smear was briefly washed with tap water and blot dry
10. The slides were examined under the microscope
Expected results

Both of these microorganisms are from two different groups. Therefore there are two
expected result are in this gram staining experiment. Gram positive slide will shows purple
colour while gram negative slide will shows pink-red colour.