SBL 1023

Technique in Biology And

Biochemistry Laboratory

Lab 5: microbiology

(gram staining and aseptic technique)

NOR AIYNA SYERA BT MOHD ASRI

STUDENT`S NAME
( E20161015981)

GROUP B

DATE/DAY/PLACE 21/11/2017 /TUESDAY/ B2-L3-MP 14

LECTURE`S NAME Professor Madya Dr. Shakinaz Binti Desa

 Part A: Gram Staining

Title

- Gram staining

Objective

- To differentiate between the two major categories of bacteria: Gram positive and
Gram negative.
- To understand how the Gram stain reaction affects Gram positive and Gram negative
bacteria based on the biochemical and structural differences of their cell walls.
- To determine the Gram stain of the bacterial sample.
- Knowledge of how distinguish the gram-positive and gram-negative bacteria properly.

Introduction

Gram staining is a method commonly used to determine the chemical make-up of the
cell wall of bacteria. The cell wall can stain either positive or negative, depending on its
chemistry. Knowing the chemical make-up makes it easier to manipulate the bacteria for
various purposes. The medical, research, developmental, and biotechnical fields are just a few
occupations whose greatest scientific advancements were due to knowing the chemical
composition of the bacteria cell wall. If the bacteria stains positive it will retain a purple/blue
colour. If the bacteria stains negative, the bacteria will not retain the purple/blue colour, but
rather have a pinkish/red colour.

Gram staining is a common technique used to differentiate two large groups of bacteria
based on their different cell wall constituents. The Gram stain procedure distinguishes
between Gram positive and Gram negative groups by colouring these cells red or violet.
Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in
their cell walls, which retains the crystal violet these cells are stained with. Alternatively,
Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which
does not retain the crystal violet during the decolouring process.
 Gram staining involves three processes: staining with a water-soluble dye called crystal
violet, decolorization, and counterstaining, usually with safanin. Due to differences in the
thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram
negative bacteria, Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal
violet stain during the decolorization process, while Gram negative bacteria lose the crystal
violet stain and are instead stained by the safranin in the final staining process.

There are several objectives why we should do this experiment such as knowledge of the
differences between gram positive and gram negative bacteria, develop the lab skill, and be
more familiar with gram staining procedure. There are also several materials that we use in
this experiment such as Hucker’s crystal violet, Gram’s iodine, 95 % alcohol, safranin and
many others. Before that, we absolutely must know about the proper procedures during this
experiment such as prepare smears from culture of microorganisms, heat the smears, place
the slide on a staining rack and so on.

Material & Reagent

- Slit
- Tube holder
- Bunsen burner
- Microscope under 100x magnification
- Inoculating loop
- Slide
- Reagent
- Distilled water
- Crystal Violet
- Gram Iodine
- Ethanol
- Safranin
Methodology

1. 1 drop of sterile water was added to the slide used a sterile inoculating loop. While, a
smear of Staphylococcus aureus was prepared.
2. The sterile water was air dry and heat fix.
3. The smear was covered with Crystal Violet (primary stain) for 1 min.
4. The slide was gently washed with water.
5. Gram’s Iodine (mordant) was added and covered for 1 min.
6. Then, the slide was washed with water.
7. Decolorized with 95% ethanol. As soon as the purple colour has stopped leaching off
the slide, decolorized the alcohol. Then, immediately washed with water.
8. The smear was covered with Safranin for 30 seconds.
9. Both the top & the bottom of the slide was washed with water.
10. The slide was blotted.
11. The smear was viewed up to 100x with immersion oil used the light microscope.

Result

Type of microbe
Observation
(under 100x oil immersion)
Gram-positive cell
Stain dark purple due to retaining the
primary dye called Crystal Violet in
the cell wall.

gram staining of Staphylococcus aureus

Gram-negative cell

Stain pink due to retaining the counter

staining dye called Safranin.

Gram staining of Escherichia coli

Discussion

In this experiment, we focused on gram staining and the mechanisms in gram staining.
As usual, before we started the experiment, it is crucial for us to perform aseptic technique In
order to minimize contamination. Firstly, we prepared smears of two microorganisms which
are Bacillus anthracis and Escherichia coli. In theory, both of these microorganisms are from
two different groups, and we expected to observe two different results in this gram staining
experiment. After preparing the smears of the microorganisms, the smears were air dried and
heat fixed. Then we stain both smears using crystal violet dyes. Crystal violet dyes are basic
dyes that have positively charged particle that helps them to bind to negatively charged
molecule like teichoic acid at the cell wall of bacteria.

This crystal violet dye can dissociate into cv+ and cv- ions. These ions can penetrate
deeps into the cell wall of bacteria and interacts with the negatively component on the
bacterial cell wall. 1 minute later, the crystal violet was washed with tap water and then the
slides are dried. The next step is to add iodine onto each smears. Iodine was being added as a
mordent to form crystal violet-iodine complex, CVI complex. This complex enables the dyes
to not be easily being removed. Next, we washed the iodine with tap water and dried off the
excess water. After that, 95% of ethyl alcohol was being added to acts as a decolorizing
agent. It interacts with the lipid membrane of both positive and negative bacteria, and this
would cause the gram-negative bacteria to lost their outer membrane and exposing the
peptidoglycan. The CVI complex are being washed from the outer membrane of gram-
negative bacteria and cause the purple colour to decolorize.

Meanwhile, for gram-positive bacteria, the addition of alcohol dehydrated the layer of
peptidoglycan which in turn would trap the CVI complex. This cause the gram positive
bacteria appeared to be purple colour as the CVI complex are being retained. The addition of
alcohol is not be more than 15 seconds as this would break the cell wall of the bacteria, thus
resulting in no stain to be observed. The slides are washed with water and dried off. In the
next step, safranin was used to counterstain both smears. This is to enables the gram negative
bacteria to be visualized easily as it can be stain in pink colour. The gram-positive bacteria
does not being stained pink when safranin was being introduced because the peptidoglycan
layer already have CVI complex. Then, the slides were washed using tap water and dried off.
Finally, we observed our specimen using microscope under oil-immersion objective lenses.
This particular lens has more mirrors inside and it requires the use of oil to refract light rays
towards the center of the lenses.

Conclusion

For the experiment, in the conclusion, gram staining have the different between the positive
and negative. The gram positive bacteria stain give a purple colour. Meanwhile the gram
negative bacteria give pink colour.

References

1. Monica Z. Bruckner, gram staining, 2017, Retrieved from,

https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html
on 25/12/17.
2. Wikipedia, gram stain, 2017, Retrieved from,
https://en.wikipedia.org/wiki/Gram_stain on 25/12/2017.
 Part B: aseptic techniques

Title

- Aseptic techniques

Objectives

- To acquire the skill of aseptic technique in the field of Microbiology.

- To prevent contamination of cultures and media from microbes in the environment.
- To transfer cultures from one medium by inoculating another medium. This is called
subculturing.
- To isolate a microorganism from a mixed culture to obtain a pure culture.
- To prevent lab microorganisms from being spread in the environment and/or infecting
the investigator.

Introduction

Aseptic technique is a fundamental and important laboratory skill in the field of

microbiology. Microbiologists use aseptic technique for a variety of procedures such as
transferring cultures, inoculating media, isolation of pure cultures, and for performing
microbiological tests. Proper aseptic technique prevents contamination of cultures from
foreign bacteria inherent in the environment. For example, airborne microorganisms
(including fungi), microbes picked up from the researcher’s body, the lab bench-top or other
surfaces, microbes found in dust, as well as microbes found on unsterilized glassware and
equipment, etc. may potentially contaminate cultures, thus interfering with the lab results.
Using proper aseptic technique can greatly minimize or even eliminate the risk of
contamination. In addition, aseptic technique is of utmost importance to maintain pure stock
cultures while transferring cultures to new media. Aseptic technique is also essential for
isolation of a single species of microorganism from a mixed culture to obtain a pure culture.
Furthermore, proper aseptic technique prevents microbes used in the laboratory from
accidentally being released into the environment and/ or infecting people working in the
laboratory. This is especially relevant when pathogens are being handled.
Material and reagent

- A stock culture of E.coli

- Inoculation loop
- A striker/lighter
- Bunsen burner,
- Ethanol
- Agar plates
- Paper towels
- Water
- Hand sanitizer
- Soap

Methodology

(A) Streak plate technique

1. The inoculating loop was sterilized on the Bunsen burner by putted the loop into the
flame until it was red hot. Allowed it to cool.
2. An isolated colony was collected from the agar plate culture of (a) E. coli and and spread
each of them over the first quadrant on separate agar plate.
3. The agar plate was covered with the lid and flame the loop.
4. Turned the plate and lightly streaked into the next quadrant without overlapping the
previous streak.
5. Repeated step 3 and 4 and streaked into the third quadrant.
6. Sealed each plate with Parafilm.
7. Inverted the plates and incubate at 37oC for 24 hours.

(B) Effect of handwashing on bacteria on thumb

1. 1.4 nutrient agar was obtained and labelled them as:

a) Control
b) Water
c) Hand sanitizer
d) Soap
 2. Each agar plate was divided into 4 sections by drawing line used a marker pen on the
back of the petri dish.
3. Gently pressed the thumb on the control agar plate used aseptic technique
4. The hands was washed with water and repeated step 3 on the appropriate agar.
5. Step 4 was repeated using hand sanitizer and soap.
6. Each plate was sealed with Parafilm.
7. The plates has been inverted and incubated at 37oC for 24 hours.

Result

Plate image

Plate A , Streak plate technique

Plate B , Effect of handwashing on bacteria

on thumb
Discussion

Aseptic technique aims to prevent pathogenic organisms, in sufficient quantity to cause

infection, from being introduced to susceptible body sites by the hands of staff, surfaces
or equipment. It protects patient during invasive clinical procedures by utilising infection
prevention measures that minimise the presence of micro-organisms. Aseptic transfer is
transferring living microbes from one place to another without contamination of the culture,
the sterile medium, or surroundings.

In addition, aseptic technique is of utmost importance to maintain pure stock cultures

while transferring cultures to new media. Aseptic technique is also essential for isolation of a
single species of microorganism from a mixed culture to obtain a pure culture. Furthermore,
proper aseptic technique prevents microbes used in the laboratory from accidentally being
released into the environment and/or infecting people working in the laboratory. This is
especially relevant when pathogens are being handled

Conclusion

Aseptic technique is a fundamental and important laboratory skill in the field of

microbiology. Microbiologists use aseptic technique for a variety of procedures such as
transferring cultures, inoculating media, isolation of pure cultures, and for performing
microbiological tests. Proper aseptic technique prevents contamination of cultures from
foreign bacteria inherent in the environment. For example, airborne microorganisms
(including fungi), microbes picked up from the researcher’s body, the lab bench-top or other
surfaces, microbes found in dust, as well as microbes found on unsterilized glassware and
equipment, etc. may potentially contaminate cultures, thus interfering with the lab results.
Using proper aseptic technique can greatly minimize or even eliminate the risk of
contamination.

References

1. Microbiological technique – the basics. Retrieved from,

http://www.biotopics.co.uk/microbes/tech%.html on 20/12/2017.
2. Welcome to Microbugz – Aseptic techniques. Retrieved from,
http://www.austincc.edu/microbugz/aseptic-technique.php on 20/12/2017.