Name: Altynshash Akhmetova

Course number: PED 305

Lab Section: 05-L

Date: 27.02.2023

LAB REPORT

TITLE: DETERMINATION OF SPECIES OF BACTERIA IN “LACTEL” MILK,

BY USING GRAM STAINING TECHNIQUE.

Introduction:

The Gram staining method is named after the Danish bacteriologist Hans Christian Gram (1853-
1938) who originally devised it in 1882 (but published in 1884), to discriminate between
pneumococci and Klebsiella pneumoniae bacteria in lung tissue. Gram was searching for a
method that would allow visualization of cocci in tissue sections of lungs of those who had died
of pneumonia. The Gram stain is a differential staining method of differentiating bacterial
species into two large groups: Gram-positive and Gram-negative bacteria, based on the chemical
and physical properties of their cell walls. It is a very important staining method in the initial
characterization and classification of bacteria. Gram-positive bacteria have cell walls that contain
thick layers of peptidoglycan (90% of cell wall). These stain purple. Gram-negative bacteria
have walls with thin layers of peptidoglycan (10% of wall), and high lipid content.

Addition of negatively charged iodine (in the mordant) binds to the positively charged dye and
forms a large dye-iodine complex within the cell. Crystal violet interacts with aqueous KI-I2 via
a simple anion exchange to produce a chemical precipitate. During decolorization, alcohol
dissolves the lipid present in the outer membrane of Gram negative bacteria and it leaches the
dye-iodine complex out of the cell. A thin layer of peptidoglycan does not offer much resistance
either. The dye-iodine complexes are washed from the Gram negative cell along with the outer
membrane. Hence, Gram negative cells readily get decolorized. On the other hand, Gram
positive cells become dehydrated from the ethanol treatment, closing the pores as the cell wall
shrinks during dehydration. The dye-iodine complex gets trapped inside the thick peptidoglycan
layer and does not get decolorized.

Purpose and objectives of the experiment: to differentiate the bacterial species in the “Lactel”
milk, to identify gram-positive and gram-negative bacteria and to study the morphological
structure of lactococci.

Hypothesis: The bacteria in the milk will be stain purple after the whole procedure of gram
staining, since lactococci are defined as gram-positive bacteria.

Materials and Methods:

The gram staining method has two stages: preparation of a bacterial smear and staining a
micropreparation. For the first part of the experiment, a slide, an inoculating loop, a Petri dish
with bacteria that grown in necessary agar media and a Bunsen burner for thermal fixation is
required. For the second part, materials were used, such as a primary stain (crystal violet),
mordant (Iodine), ethanol alcoholacetone as a decolorizer and fuchsin as a counterstain.
Furthermore, three beakers with distilled water for washing and dispensers with tips were used.
The experiment was carried out in sterile conditions, with a lab coat and gloves.

Smear preparation and thermal fixation: When preparing a sample for staining attention should
be paid to the thickness of the smear and it should not be too thick. First of all, using a sterile
inoculating loop, it is necessary to add 1 drop of sterile water to the slide. Secondly, a sample of
the bacteria should be transferred to the slide also with an sterile inoculating loop. Then, the
sample transferred should be smeared on the slide and left to dry in the air. Lastly, it is thermal
fixation; we need to carefully pass the slide with dried sample, through the Bunsen burner.

Staining Procedure: In the first step, we need to apply crystal violet reagent to the slide and
leave it for 1 minute. After that, it is necessary to carefully rinse the slide with water. In the
second step, we need to add iodine mordant into the slide and wait for 1 minute. Then, rinse the
slide again with distilled water. In the third step, we need to fill the slide with a decolorizing
agent, namely ethanol (95%) and wait for 15 seconds. At the final step of the staining, we add
counterstaining solution, in this case a fuchsin was used. After 30 seconds, we need to rinse the
slide again and leave it to dry. After all these steps, we can examine the smear under the
microscope with lenses of different magnifications. When using a 100x lens, it is necessary to
add a drop of immersion oil to the stained sample.

Results:

As a result of this experiment, both types of bacteria were observed. It stain purple and pink. The
most of the bacteria were purple, which proves the gram-positive nature of lactococci. In
addition, there were gram-negative bacteria under the microscope, we determined them
according to pink stain. Thus, we can conclude that gram staining method helps to differentiate
bacteria into two types. However, in the milk sample, we were able to see both gram-positive
lactococci and gram-negative bacteria, which probably got into the Petri dish because of
contamination from the environment.
Discussion:

1. Gram-positive bacteria are colored purple, and gram-negative bacteria are colored red or pink.
Gram-positive cells have a thick layer of peptidoglycan in the cell wall and it retains crystalline
violet dye. Gram-negative cells have a thinner layer of peptidoglycan, which allows the dye to be
washed out with ethanol. When looking the smear under microscope, it was concluded that most
of the bacteria were purple, which showed that they are gram-positive bacteria. However, pink-
colored bacteria have also been obtained and this allows to conclude that the milk also contains
gram-negative bacteria.

2. It was expected to see only gram-positive bacteria in the milk under the microscope, since
lactococci are gram-positive. However, as it mentioned before gram-negative bacteria is also
noticed and it was unexpected. One of the factors of it can be poor quality of the smear
preparation. Also, excessive decolorization of sample can lead to the detection of false gram-
negative results and insufficient decolorization can lead to the detection of false gram-positive
results.

3. The hypothesis is not always 100% correct, so sometimes unexpected results of the
experiment may occur because of some errors during the experiment. The hypothesis at the
beginning of the experiment was tested and can be partially accepted. According to the
hypothesis, the bacteria in the milk will be colored purple after gram staining, since lactococci
are defined as gram-positive anaerobic bacteria. some part was colored pink, which indicates the
presence of gram-negative bacteria in the milk. One of the possible reasons for partial accepting
of the hypothesis is that some part was colored pink, which indicates the presence of gram-
negative bacteria in the milk. Overall, the hypothesis has been proven, without taking into
account the unexpected results.