Lab 1: Aseptic Technique (Hand Washing

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Lab 1: Aseptic Technique (Hand Washing)
Introduction
When working with microorganisms it is desirable to work with a pure culture. A pure culture
is composed of only one kind microorganism. Occasionally a mixed culture is used. In a
mixed culture there are two or more organisms that have distinct characteristics and can be
separated easily. In either situation the organisms can be identified. When unwanted
organisms are introduced into the culture they are known as contaminants.
Aseptic technique is a method that prevents the introduction of unwanted organisms
into an environment. Aseptic technique is used to prevent possible infection. When working
with microbial cultures aseptic technique is used to prevent introducing additional organisms
into the culture.
Handwashing is thought to be effective for the prevention of transmission of diarrhoea
pathogens. However it is not conclusive that handwashing with soap is more effective at
reducing contamination with bacteria associated with diarrhoea than using water only. In this
study 20 volunteers contaminated their hands deliberately by touching door handles and
railings in public spaces. They were then allocated at random to (1) handwashing with water,
(2) handwashing with non-antibacterial soap and (3) no handwashing. Each volunteer
underwent this procedure 24 times, yielding 480 samples overall. Bacteria of potential faecal
origin (mostly Enterococcus and Enterobacter spp.) were found after no handwashing in 44%
of samples. Handwashing with water alone reduced the presence of bacteria to 23% (p <
0.001). Handwashing with plain soap and water reduced the presence of bacteria to 8%
(comparison of both handwashing arms: p < 0.001). The effect did not appear to depend on
the bacteria species. Handwashing with non-antibacterial soap and water is more effective for
the removal of bacteria of potential faecal origin from hands than handwashing with water
alone and should therefore be more useful for the prevention of transmission of diarrhoeal
diseases.
An aseptic technique experiment was conducted to test the effects of aseptic technique
on the transfer of target bacteria, Sarcina aurantiaca (S. aurantiaca). S. aurantiaca was
transferred from stock plates to treatment plates using aseptic techniques and three improper
variations of aseptic techniques to test the impact of aseptic techniques on contamination
growth in culture plates. The hypothesis for this experiment was that cultures inoculated with

Lab 1: Aseptic Technique (Hand Washing)

S. aurantiaca using aseptic technique will have less growth of biological contaminants than
cultures inoculated using a non-aseptic technique. The null hypothesis was that plates
inoculated using aseptic technique will have the same amount of contamination as plates
inoculated using non-aseptic technique.

Objectives
1. To learn, understand, and practice aseptic transfer techniques.
2. To examine validity of hand washing as a limit to disease causing agent.

Methods
1. Obtain a nutrient alga plate.
2. Turn the place upside down and use a wax pencil or Sharpie to make a graphic that
divides the plate into 5 sections. Label the sections-1, 2, 3, 4, 5. The numbers will
correlate with the following actions: 1. Unwashed; 2. Water rinse; 3. Soap and water;
4. Brush, soap and water; 5. Sanitizer.
3. On #1 lightly stroke a finger across (inoculate) the media. Rinse your hands briefly
with water, flick off excess water, and inoculate #2. Wash your hands with soap and
water, flick off excess water – inoculate #3. Use the brushes provided and scrub your
hands well, pat dry, and inoculate #4. Last of all, rub your hands thoroughly with the
alcohol- based hand sanitizer, allow to air dry, then inoculate #5.
4. Incubate the plates for 48 hours at 35-37oC.
5. Hypothesize which section of the plate will have the most growth, which will have the
least.
6. Evaluate the growth on each plate and check the hypotheses made. Record all
findings.

Lab 1: Aseptic Technique (Hand Washing)

Results
Thumbprint
Part

Percentage of bacteria attached in alga
plate after
2 days (%)

A Unwash

25

B Water Rinse

20

C

10

Brush, soap and water
D
Sanitizer

Observation

30

Lab 1: Aseptic Technique (Hand Washing)

Discussion
In this experiment, my group and I have conduct an experiment about aseptic technique on
hand washing and feet. For aseptic technique on hand is divided into four categorize which is
the hand is unwash, rinse by water, using sanitizer and brush, soap and water. Next
experiment is use aseptic technique on feet which has been categorize into 4 section with their
right feet of difference students. We observed the growth of the bacteria in agar plate of both
experiment for three days and recorded the data in term of percentage the bacteria grown.
Firstly, aseptic technique on hand have shown the result on the second day that the
highest bacteria growth in agar plate is by using sanitizer which is 30 % and the least is by
using brush, soap and water wthin10 %.

For unwash hand and rinse with water the

percentage is within 25% and 20% respectively on second day. This prove that bacteria still
there in our hand although we have wash it with water, soap, brush and sanitizer. As we all
known that soap and sanitizer can reduce the growth of the bacteria in our hands. This can be
prove that only 10 % of the bacteria growth on the agar plate. Soap molecules contain both
hydrophilic (likes water and goes into solution) and hydrophobic (repels water). Bacteria (and
dirt and oils) get bound to the hydrophobic end of the soap molecules and the hydrophilic end
helps it get washed away with water. This is just basic soap with no antibacterials in it.
Furthermore, by using sanitizer the percentage of the bacteria growth must be
decrease because the alcohol in that use for sanitizers can dehydrates bacteria, unfolds their
proteins, and kills them. The result may be happen due to the plates has been left open during
inoculation because bacteria present in the air and other surfaces could have contaminated the
culture producing incorrect experimental data. Although great care was taken to avoid
contamination of the sterile agar plates, our lab partner let his hand to dried at the
environment or his hands accidentally touched the object that cause the hand been
contaminated prior to inoculating the agar plate. This could have been the cause for more
bacteria than expected on the sanitizer agar plate. Rinsed the hand with water also can reduce
the bacteria growth but it is not the effective ways because as we can see the result compared
with unwash hands the growth of the bacteria decrease about five percent in agar plate on
second day.

Lab 1: Aseptic Technique (Hand Washing)

On the third day, the percentage growth of the bacteria increased on the agar plate that
means that the bacteria had rapidly duplicated. The highest percentage of the bacteria growth
is by using soap, brush and water within 80% and the lowest is by water rinse which is 35%.
For the next experiment, the percentage growth bacteria by using method of the
aseptic technique on feet shows that the highest percentage of the region of bacteria growth
on agar plate is Khairy’s feet within 40% than the other members on the second day. The least
percentage of bacteria growth on the second day is Asyraf’s feet within 10% and for John and
Azrim feet the percentage for both is the same. This is prove that the bacteria can attach on
our skin even though it has been covered. On the third days, the percentage of the bacteria
growth increases because the bacteria can duplicate itself to produce more bacteria of their
kinds.

Questions:
1. What is agar ?
An agar plate is a Petri dish that contains a growth medium (typically agar plus nutrients)
used to culture microorganisms or small plants like the moss Physcomitrella patens. Agar is a
polymer made up of subunits of the sugar galactose, and is a component of the cell walls of
several species of red algae that are usually harvested in eastern Asia and California.
Dissolved in boiling water and cooled in a petri dish, laboratory agar looks gelatinous.
Although agar's chief use is as a culture medium for various microorganisms, particularly for
bacteria, its other less well-known uses include serving as a thickening for soups and sauces,
in jellies and ice cream also in cosmetics, for clarifying beverages and for sizing fabrics.
Furthermore, agar unlike gelatin, it won't be degraded (eaten) by bacteria. Also, agar is
firmer and stronger than gelatin. It's still possible, however, to use gelatin as a culture medium
for bacteria if agar is unavailable. Agar polysaccharides serve as the primary structural
support for the algae's cell walls.
2. Do you know the compositions needed in making an agar growth media for
bacteria?
The composition of making an agar growth media are;
 0.5% Peptone

Lab 1: Aseptic Technique (Hand Washing)

It is an enzymatic digest of animal protein. Peptone is the principal source of organic
nitrogen for the growing bacteria.
 0.3% beef extract/yeast extract
It is the water-soluble substances which aid in bacterial growth, such as vitamins,
carbohydrates, organic nitrogen compounds and salts.
 1.5% agar
It is the solidifying agent.
 0.5% NaCl
The presence of sodium chloride in nutrient agar maintains a salt concentration in the
medium that is similar to the cytoplasm of the microorganisms.
 Distilled water
Water is essential for the growth of and reproduction of micro-organisms and also provides
the medium through which various nutrients can be transported.
 pH is adjusted to neutral (7.4) at 25 °C.

3. Provide three reasons why the use of aseptic technique is essential when handling
microbial cultures in the lab?
This is because 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.
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.

4. Where and how should a label be written on an agar plate? What about on a test
tube?

Lab 1: Aseptic Technique (Hand Washing)

Petri dishes are labelled on the bottom rather than on the lid. Write close to the edge of
the bottom of the plate to preserve area to observe the plate after it has incubated. Labels
usually include the organism name, type of agar, date, and the plater's name or initials. Using
sterile cotton swabs, remove any visible water on the agar in the plate or around the inner rim
of the petri plate. Observe the plate and mentally divide it into four sectors. The plate will
then be turned clockwise (if you are right handed) with the agar side up. The second sector
will then be at the top for streaking and then the plate is turned again so that the third and four
sector can be streaked.
For labelling the test tube do not label tubes on the cap and labels usually include the
organism name, type of agar, date, and the tubes's name or initials.

5. How should agar plates be incubated? Why?
Agar plates should be incubated in an inverted position to prevent condensation on the
agar surface that could spread the inoculated organisms. Incubating the plates to promote
growth of microbes is an essential part of any microbiology investigation. Incubating in
aerobic conditions, and below human body temperature, reduce the risk of encouraging
microorganisms (particularly bacteria) that could be pathogenic to humans.

Conclusion
In conclusion, aseptic technique is a very useful method on reducing the growth of the
bacteria. It has been prove that using the soap, brush and sanitizer can reduce the growth of
the bacteria in our hand. Aseptic technique is a basic laboratory technique that must be
employed especially during Microbiology laboratory session so as to prevent any
contamination and affecting the accuracy of the result. Since microorganism can replicated
rapidly, disposal of contaminants must be done properly so as to protect both the equipments
and the health of individuals. It is highly recommended to use aseptic technique to perform
such microbiological work in order to enhance accuracy in findings.

Lab 1: Aseptic Technique (Hand Washing)

Reflection
From this experiment, I learned to understand and practice aseptic transfer techniques. We
used aseptic technique when to prevent possible infection and of unwanted organisms into an
environment and culture. Next, I learned how to examine validity of hand washing as a limit
to disease causing agent that obtain a nutrient alga plate. After doing this experiment, I
realize that aseptic technique is very important for us in each activities. Aseptic technique is
a method designed to prevent contamination from microorganisms. With this technique, we
may minimize the risks that we'll experience an infection while we doing on activities with
microbes or bacteria. So here I can conclude that the aseptic technique is very important in
each experiment.
References
Bettelheim, Frederick A. (2010). Laboratory experiments for introduction to general, organic
and biochemistry 7th ed. Brooke-Cole.
Bres, M. & Weisshaar, A. (2012). Thinking About Biology. An Introductory
Laboratory Manual. 4th Edition. Benjamin-Cummings Publishing.
Brown, Alfred E. (2012). Benson’s microbiological applications. Laboratory manual
th

in General microbiology. 12 edition. McGraw Hill.
Gallagher, S.R. & Wiley, E.A. (2012). Current Protocols Essential Laboratory
Techniques. 2nd edition. Wiley Blackwell
Mader, Sylvia S. (2012). Laboratory manual essentials of biology. McGraw Hill
Wilson, K. & Walker, J. (2010). Principles and techniques of biochemistry and
molecular Biology. 7th edition. Cambridge University Press, New York.

Lab 1: Aseptic Technique (Hand Washing)

Lab_2 Gram Staining

Lab 2: Gram Staining

Introduction
Gram staining is not a simple stain, but rather is known as a differential technique. A
differential technique is a process that distinguishes between a variety of microbial organisms
based on the ability of their cell wall to hold certain dyes. The Gram staining technique
depends upon the ability of a microbial cell wall to resist decolorization.
Most common bacteria are either Gram-positive or Gram-negative (based on cell wall
structure). Remember from lecture, Gram-positive cell walls consist of several layers of
peptidoglycan (cross-linked by teichoic acid and lipoteichoic acid). Gram-negative cell walls
have one layer of peptidoglycan surrounded by a lipid-based outer membrane. In the 1880s,
Hans Gram developed the differential method of staining that bears his name. While we still
don’t know exactly why Gram-positive bacteria end up looking different from Gram-negative
bacteria, Gram-staining is still an important way to characterize bacteria.
Gram-staining begins by getting cells to stick on a clean microscope slide. Such a prep is
called a bacterial smear. To a bacterial smear the following chemicals are applied to make a
Gram-stain:
1. Gram’s Crystal Violet Stain
The smear is flooded with Crystal Violet for 60 seconds. Crystal violet is a purple chemical
that sticks to the peptidoglycan layer of the bacterial cell wall. After 60 seconds, crystal violet
is rinsed off using distilled water.

2. Iodine
The smear is next flooded with Iodine for 60 seconds. The iodine is called a mordant—it
causes crystal violet to stick to peptidoglycan like mortar causes bricks to stick together. After
60 seconds, iodine is rinsed off using distilled water.

3. Acetone/Ethanol Wash
The smear is next flooded with Acetone/Alcohol for JUST 5 SECONDS. The
Acetone/Alcohol washes crystal violet out of the Gram-negative cell wall. We’re not really

Lab_2 Gram Staining

sure why this happens, but it does. The Gram-positive cell wall retains crystal violet as long
as the acetone/alcohol wash lasts not more than a few seconds. After 5 SECONDS, the
acetone/alcohol is rinsed off using distilled water. The acetone/alcohol wash is the differential
step in the Gram-stain process. That is, it is the acetone/alcohol that creates the observable
difference: Gram-positive cells look purple after this step; Gram-negative cells look clear.

4. Safranin Stain
The smear is flooded with Safranin for 90 seconds to two minutes. Safranin is a pink stain
that sticks to cytoplasmic components of the cell. All cells become stained with Safranin.
Grampositive cells are pink on the inside, but you can’t see this because they are dark purple
on the outside (kind of like a bon-bon). Gram negative cells, which were cleared in the
previous step, end up looking pink. After 90 seconds to two minutes (the longer the better),
Safranin is rinsed off with distilled water.

5. Drying the slide
The completed Gram-stained slide is stuck into a book of bibulous paper and allowed to dry
for a minute or two. Once dry, the slide is ready for observation.
Gram-positive cell walls have a thick peptidoglycan layer beyond the plasma
membrane. Characteristic polymers called teichoic and lipoteichoic acids stick out above the
peptidoglycan and it is because of their negative charge that the cell wall is overall negative.
These acids are also very important in the body’s ability to recognize foreign bacteria.
Grampositive cell walls stain blue/purple with the Gram stain.
Gram-negative cell walls are more complex. They have a thin peptidoglycan layer and
an outer membrane beyond the plasma membrane. The space between the plasma membrane
and the outer membrane is called the periplasmic space. The outer leaflet of the outer
membrane is composed largely of a molecule called lipopolysaccharide (LPS). LPS is an
endotoxin that is important in triggering the body’s immune response and contributing to the
overall negative charge of the cell. Spanning the outer membrane are porin proteins that
enable the passage of small molecules. Lipoproteins join the outer membrane and the thin
peptidoglycan layer.
Gram-negative cells will stain pink with the Gram stain.

Lab_2 Gram Staining

Objectives
1. To prepare and interpret a Gram stain.
2. To identify primary stain, mordant, decolorizer, and counterstain procedures in gram
staining.
3. To identify organisms as gram-positive or gram-negative.

Materials
TSB tube of organism #1(E.coli or Klebsiella), TSB tube of organism #2 (Staphylococcus
epidermidis or Streptococcus bovis), TSB tube of organism #3 (S. aureus), Microscope slides,
Gram’s staining set including: 1. Gram’s crystal violet, 2. Gram’s iodine, 3. Decolorizer (95%
ethyl alcohol), 4. Safranin, Inoculating loop and needle, Bunsen burner, Immersion oil and
Light microscope.

Methods
1. Make a thin layer smear on a clean glass slide, dry it in air and fix by passing
through flame of a burner.
2. Cover the smear with crystal violet, keep it for one minutes.
3. Wash the slide with water, then cover with Gram iodine and let it stand for one
minute.
4. Wash the slide with water.
5. Decolour with acetone/ alcohol, rocking the slide gently for 10-15 seconds till the
violet colour comes off the slide.
6. Wash with water immediately.
7. Counterstain with safranin. Let the counterstain stand for 30 seconds.
8. Wash with water, blot dry.
9. Place under the microscope for observation, adjust the scope accordingly and,
using the oil immersion lens for viewing, draw/ describe what is observed on the
report.
Results

Lab_2 Gram Staining

Gram Stain
Procedure

Reagent

Cell Color
Gram Positive

Gram Negative

Fixed cells on slide

-

Colourless

Colourless

Primary stain

Crystal Violet

Purple

Purple

Mordant

Iodine

Purple

Purple

Decolorizer

Alcohol

Purple

Colourless

Counterstain

Safranin

Purple

Red

An easy way to remember the steps of the Gram stain is Come In And Stain !
Come – Crystal Violet
In

- Iodine

And

- Alcohol

Stain - Safranin

Lab_2 Gram Staining

Observation

E.coli at focus 40times (40X) in light microscope

S.aureus at focus 40times (40X) in light microscope

Lab_2 Gram Staining

Discussion
In this experiment, the main objective is to prepare and interpret a Grain stain. The Gram
staining is a type of microbiology or laboratory experiment that determines whether bacteria
are present. It also determines whether bacteria are gram negative or gram positive. The
difference between gram negative and gram positive bacteria can be important when
determining appropriate treatment for an infection. Gram stains are performed on various
types of specimens including blood, tissue, stool, and sputum. In some instances, it is not
clear whether an infection is bacterial, fungal, or viral. And these types of infections may be
treated very differently. In addition, different types of bacteria may require different
treatments. A gram stain lets the physicians to determine whether bacteria are causing an
infection and what type of bacteria is present in the infections.
Next, the second objective of the experiment is to identify the primary stain, mordant,
decolorizer and the counterstain procedures in the gram staining. The primary stain is used in
Gram staining to detect Gram-positive bacteria. The primary stain is a crystal violet color.
The application of the primary stain is to heat-fixed smear of a bacterial culture. Heat fixation
kills some bacteria but is mostly used to affix the bacteria to the slide so that they don't rinse
out during the staining procedure. The mordant is classically defined as an ion that binds a
chemical dye and holds it down, such that the dye remains stuck on the organism. In this
experiment the mordant is the Gram iodine that is added after the crystal violet. The purpose
of the mordant is to fix the crystal violet to the bacterial cell wall. The decolizer in this
experiment is acetone. Decolorizing the cell causes this thick cell wall to dehydrate and
shrink which closes the pores in the cell wall and prevents the stain from exiting the cell.
Lastly, the counterstain, which is usually positively charged safranin or basic fuchsine, is
applied last to give decolorized gramnegative bacteria a pink or red color.
The last objective of this experiment is to identify the organisms as gram-positive or gramnegative. Gram-positive organisms generally have a single membrane surrounded by a thick
peptidoglycan. Gram-positive organisms take up the crystal violet stain used in the test, and
then appear to be purple-coloured when seen through a microscope. This is because the thick
peptidoglycan layer in the bacterial cell wall retains the stain after it is washed away from the
rest of the sample, in the decolorization stage of the test. The gram-negative organisms
generally possess a thin layer of peptidoglycan between two membranes. Gram-negative
organisms are a group of bacteria that do not retain the crystal violet stain used in the Gram
staining method of bacterial differentiation. After staining with crystal violet, an alcohol wash
is applied which decolorizes the bacteria showing that their peptidoglycan layer is too thin to

Lab_2 Gram Staining

retain the stain and enabling identification. A counterstain is then added which recolorizes the
bacteria red or pink.

Questions
1. How does cell wall different from the plasma membrane?
Cell wall is the outermost boundary in plant cell whereas plasma membrane or
cell membrane is the outermost boundary in the animal cells. In a plant cell, cell
membrane is found in the inner side of the cell wall. Cell membrane is present in all
cells include animal cell and plant cell. Cell wall is absent in animal cells.
Cell wall is rigid, thick structure and visible in light microscope. Cell membrane is
delicate and thin structure and only visible in electron microscope. Cell wall is
completely permeable to ordinary macromolecule. Cell membrane is selective
permeable or semipermeable allowing only certain molecule to pass through.
Cell wall is metabolically inactive and non-living. Cell membrane is metabolically
active and living. Cell wall is made up of cellulose in plant cell wall.
Plasma membrane is made up of lipids proteins and small amount of carbohydrate.
2. In what ways is the cell wall difference between Gram positive and Gram negative
bacteria?
Gram positive bacteria, the cell membrane is much thicker than the gram- bacteria
containing about five times the amount of peptidoglycan and has a smooth appearance
on its external surface. It is also composed of polysaccharides and/or teichoic acids.
Gram negative bacteria, the gram-negative bacterial also have a second membrane
which is chemically different from the plasma membrane external to the cell wall, and
it also may have a gelatinous sheath external to the second membrane. The cell walls
main component is lipopolysaccharide. Additionally there is a phospholiped protein,
lipoprotein. It’s outer appearance is convoluted.
Both gram-negative and gram-positive bacteria have peptidoglycan, its physical
arrangement in the cell wall is different. In gram-positive cells the peptidoglycan is a
heavily cross-linked woven structure that encircles the cell in many layers. It is very
thick with peptidoglycan accounting for 50% of weight of cell and 90% of the weight
of the cell wall. Electron micrographs show the peptidoglycan to be 20-80 µm thick.

Lab_2 Gram Staining

In gram-negative bacteria the peptidoglycan is much thinner with only 15-20% of the
cell wall being peptidoglycan and it is only intermittently cross-linked. In both cases
peptidoglycan is not a barrier to solutes, as the openings in the mesh are large enough
for most molecules including proteins to pass through.

The gram-positive cell wall. The cell wall is made mostly of peptidoglycan,
interspersed with teichoic acid which knits the different layers together. The amount
of crosslinking is higher and the wall is thicker than in gram-negative cell walls.
Another structure in the gram-positive cell wall is teichoic acid. It is a phosphodiester
polymer of glycerol joined by phosphate groups. Amino acids such as D-alanine are
attached. Teichoic acid is covalently linked to muramic acid and stitches various
layers of the peptidoglycan mesh together. Teichoic acid stabilizes the cell wall and
makes it stronger.

Teichoic acid. Teichoic acid is a long, thin molecule that weaves through the peptidoglycan.

Lab_2 Gram Staining

The gram-negative cell wall. The cell wall in gram-negative bacteria contains
much less peptidoglycan and is surrounded by an outer membrane. There is much less
crosslinking between the peptidoglycan. LPS is also present in the outer membrane
and penetrates into the surrounding environment. The outer membrane of gramnegative bacteria is another lipid bilayer similar to the cytoplasmic membrane, and
contains lipids, proteins, and also lipopolysaccharides (LPS). The membrane has
distinctive sides, with the side that faces the outside containing all the LPS. LPS is
composed of two parts: Lipid A and the polysaccharide chain that reaches out into the
environment. Lipid A is a derivative of two NAG units with up to 7 hydrophobic fatty
acids connected to it that anchor the LPS in the membrane. Attached to Lipid A is a
conserved core polysaccharide that contains KDO, heptose, glucose and glucosamine
sugars. The rest of the polysaccharide consists of repeating sugar units and this is
called the O-antigen. The O-antigen varies among bacterial species and even among
various isolates of the same species. Many bacterial pathogens vary the make-up of
the O-antigen in an effort to avoid recognition by the host's immune system.

The structure of LPS. LPS is
composed of three sections: the
lipid A region, a conserved core
polysaccharide, and a highly
variable O-polysaccharide. (A)
The chemical structure of LPS.
(B) A molecular model of the
membrane from Pseudomonas aeruginosa. Source (T. P. Straatsma, Pacific Northwest National
Laboratory)

[A summary of the differences between gram-positive and gram-negative cell walls.]

Lab_2 Gram Staining

3. In what type of organism would the gram stain not work? Why?
Gram stain will not work with organisms that do not contain a cell wall but contains
only a cell membrane.
4. How is the Gram stain reaction by bacteria useful information to medical
doctors or microbiologists?
The information gained from gram staining is use to diagnosis, prevent and treat
bacterial infections. Gram positive Bacterial causes illness by secreting different types
of toxins that affect the cells of the infected individual. Gram negative bacteria cause
immune reactions by lipopolysaccharides found in the cell wall.

Conclusion
As the conclusion, the gram-positive organisms will form appear to be purple-coloured when
seen through a microscope and the gram-negative will form appear to be pink-coloured when
seen through a microscope. The experiment is accepted.

Reflection
In this experiment I learned how to use microscope in a proper way. I found that the bacteria
or we as microbes is very tiny and cannot view by our naked eyes. I feel happy because I can
see it by using the light microscope prepared in the lab. I feel sad also because the light
microscope cannot focus until 40 times(40X), I suggest that the lab assistant may do the
checklist for each microscope per month to ensure that the microscope is fully function.

Lab_2 Gram Staining

References
Books
Bettelheim, Frederick A. (2010). Laboratory experiments for introduction to general, organic
and biochemistry 7th ed. Brooke-Cole.
Bres, M. & Weisshaar, A. (2012). Thinking About Biology. An Introductory
Laboratory Manual. 4th Edition. Benjamin-Cummings Publishing.
Brown, Alfred E. (2012). Benson’s microbiological applications. Laboratory manual
th

in General microbiology. 12 edition. McGraw Hill.
Gallagher, S.R. & Wiley, E.A. (2012). Current Protocols Essential Laboratory
Techniques. 2nd edition. Wiley Blackwell
Mader, Sylvia S. (2012). Laboratory manual essentials of biology. McGraw Hill
Wilson, K. & Walker, J. (2010). Principles and techniques of biochemistry and
molecular Biology. 7th edition. Cambridge University Press, New York.

Internet
Gram Stain, Retrieved 09:25, January 24, 2016, from
http://web.clark.edu/tkibota/240/Lab/LM6_GramStain/GramSt
ain.pdf