Determining the morphology of bacterial species using staining

techniques and identify unknown bacterial species based on morphology

Sydney-Mae Low

Th-Eve-19

October 8, 2017
Abstract

This paper examines six different species of bacteria (Bacillus cereus, Citrobacter

freundii, Deinococcus radiodurans, Escherichia coli, Lactococcus lactis, and Providencia

rettgeri) to determine their cellular morphology. In understanding the morphology of each the

species, the results can then be used to determine whether unknown bacterial species can be

identified based of the morphology. To understand the morphology of each known bacterial

species, a series of different staining experiments were sought out using the six bacterial species

from either a solid culture or a broth culture; a simple stain experiment, a negative stain

experiment, a wet mount experiment, and a Gram staining experiment. All experiments and

organisms were viewed at 400x and/or 1000x magnification under oil immersion. This paper

seeks to answer whether unknown bacterial species can be identified using the results from the

known bacteria experiments. It is hoped that the hypothesis can either be approved or disproved

by being able to identify the unknown species or not.

Introduction

Each species of bacteria has a particular shape, arrangement, form of motility, and

cellular composition which allows for the identification of bacterial species based on their

overall cellular morphology. Different staining techniques on six different known bacteria,

Bacillus cereus, Citrobacter freundii, Deinococcus radiodurans, Escherichia coli, Lactococcus

lactis, and Providencia rettgeri, can help determine the four unknown species in the two

mixtures based on their morphology.

The purpose of a simple stain was to increase the contrast between the cells and the

background1. The six known bacteria were fixed to the slides using methanol and were stained
with crystal violet. Observation of arrangement and cell size were possible to identify using this

technique.

Negative stains, on the other hand, allow for contrast by staining the background rather

than the cells. Since slides were not fixed with methanol, the cells were less susceptible to

distortion2. This allowed observation of the nature cell size of the six bacterial species. The slides

were stained using nigrosine, an acidic stain which gets repelled by the cell wall of the bacteria.

Wet mounts do not require staining and do not fix the bacterial cells to the slide. This

made it more difficult to view the organisms but it allowed the observation of living cells.

Observation of motility, natural cell size, arrangement and shape can also be determined using

this technique3.

Gram staining also functions as a simple stain as it increases the contrast of the cells.

Gram staining, using methanol, crystal violet, Grams iodine, ethanol (ethyl alcohol, 95%), and

safranin, involved two staining procedures separated by a decolourization stage4. Gram stains

allow for the identification of Gram-positive or Gram-negative organisms (which indicated the

composition of the cell wall) by the colour of the organisms after staining.

By understanding the morphology of the six known bacteria and how they are presented

under different media, the identification of the two sets of two unknown bacteria within a

mixture can then be determined.

Methods/Experimental

The simple stain experiment protocol was established using the lab manual. The simple

stain experiment involved staining all six known bacteria; B. cereus, C. freundii, D. radiodurans,

E. coli, L. lactis, and P. rettgeri (provided by the University of Winnipeg). The process of
making a simple stain involved applying each organism (using an inoculating loop) to a drop of

saline on a slide. Saline was necessary because the organisms came from a solid culture. The

suspension of saline and bacteria was thinned out on the slide and air-dried. To fix the cells to

the slides, the slides were covered with methanol for two minutes and then rinsed with water

afterwards. To stain the cells, the bacterial smears were covered with crystal violet for

approximately two minutes (which differed from the lab manual as they called for a one minute

application of the crystal violet). The excess was then rinse with water. The slides were then

blotted with bibulous paper and observed under 400x magnification and then 1000x

magnification using oil immersion. Illustrations were made for each organism at both

magnifications.

The negative stain experiment protocol was established using the lab manual. The

negative stain experiment also involved staining all six known bacteria; B cereus, C. freundii, D.

radiodurans, E. coli, L. lactis, and P. rettgeri (provided by the University of Winnipeg). The

staining process involved starting with adding a drop nigrosine onto one end of each slide. The

bacteria were then added to the nigrosine using an inoculating loop and mixed with the nigrosine.

Using another slide at a 45 angle, the suspension was spread across the slide to form a thin

smear. After the slides had dried, the organisms were observed at 400x magnification and then

1000x magnification using oil immersion. Illustrations were made at both magnifications for all

organisms.

The wet mount experiment protocol was established using the lab manual. Similar to the

simple staining, each of the bacteria (B. cereus, C. freundii, D. radiodurans, E. coli, L. lactis, and

P. rettgeri) were added to a drop of saline on a slide and mixed. Instead of letting the organisms

dry to be stained (like the previous staining experiments), a cover slip was placed over the
mixture and was viewed at both 400x and then 1000x magnification using oil immersion.

Illustrations were made at both magnifications.

The Gram staining protocol was established using the lab manual. Gram staining

involved a more lengthy process for staining the bacteria. Using the same six known bacteria (B.

cereus, C. freundii, D. radiodurans, E. coli, L. lactis, and P. rettgeri) each organism was applied

directly onto a slide (no use for saline because each organism was cultured in a tryptic soy

broth). This step was different than directed from the lab manual as broth cultures were used

rather than solid cultures. After the smears had dried, the slides were covered with methanol for

two minutes to fix the bacteria onto the slide. The slides were then rinsed with water. The first

staining process involved applying crystal violet for a minute, followed by rinsing with water.

Grams iodine was used next to cover the slides for another minute. The excess was rinsed with

water. The decolourization stage involved removing crystal violet from Gram-negative cells

using ethanol. Ethanol was added slowly until the run-off is clear, then immediately rinsed with

water. The second staining process involved the slides being covered with safranin for

approximately 40 seconds. The slides were rinsed with water for the last time and blotted with

bibulous paper. An illustration was made for each species after viewing at 1000x magnification

using oil immersion.

The protocol for the identification of unknown bacteria was established using the lab

manual. For identification of the unknown organisms, the mixtures were prepared using the same

Gram staining technique and the slides were viewed at 1000x magnification and illustrations

were made. These organisms were then compared to the known species to see if the species

could be identified using the cell morphology.

Results

The simple stain technique made for observation of the organisms simple because of the

increased contrast. At 400x magnification, cells that were concentrated were harder to

distinguish because the cells were all darkly stained. At 1000x magnification under oil

immersion, determination of cell shapes and arrangements was also simple because it was easier

to differentiate the cells at this magnification. The morphology of each of the six bacteria is

indicated in Table 1 below:

Table 1: The cellular morphology and arrangement of the six bacterial species (Bacillus cereus,
Citrobacter freundii, Deinococcus radiodurans, Escherichia coli, Lactococcus lactis and
Providencia rettgeri) which were all grown on tryptic soy agar and prepared using a simple stain
with crystal violet and viewed at 1000x magnification with oil immersion.
Bacterial Species Cell Morphology and Arrangement
Bacillus cereus streptobacilli
Citrobacter freundii single bacilli
Deinococcus radiodurans tetrads
Escherichia coli bacilli and diplobacilli
Lactococcus lactis staphylococci
Providencia rettgeri single bacilli

Between all six bacterial species, C.

freundii was the largest in size while P. rettgeri

was the smallest and therefore the more
difficult to see, as indicated by Figure 1. L.
lactis and D. radiodurans were very
concentrated and difficult to distinguish cells at
the 400x magnification.
Figure 1: The comparison in size of A.
Citrobacter freundii which was grown on
tryptic soy agar and prepared using a simple
stain with crystal violet and viewed at 1000x
magnification with oil immersion and B.
Providencia rettgeri which was grown on
tryptic soy agar plates and prepared using a
simple stain with crystal violet and viewed at
1000x magnification with oil immersion.
 The negative stain technique did not require the cells to be

fixed (with methanol) therefore the cells were less distorted. Since

negative stain technique stains the background rather than the cells,

there appeared to be a halo-effect around each bacterial cell to

Figure 2: The individual
links (A.) in Bacillus
increase the contrast. Cells were easier to see at 400x
cereus which was grown
on a tryptic soy agar plate
magnification compared to the simple stain technique. For P.
and prepared using a
negative stain with
rettgeri there was an occasional cell that was darker than the
nigrosine and viewed at
1000x magnification with
other, possibly indicating a break in the cell wall, allowing the
oil immersion.
nigrosine to stain the cell. In observing B. cereus, it was possible to see the individual links

between the bacterial cells at 1000x

magnification, as seen in Figure 2.

The wet mount technique did not

Figure 3: The comparison of size across
preparations of A. Providencia rettgeri grown on
require the cells to be fixed (which would
tryptic soy agar and prepared using a simple
stain with crystal violet and viewed at 1000x
have resulted in the death of the
magnification with oil immersion, B.
Providencia rettgeri grown on tryptic soy agar
organisms) and did not stain them,
and prepared using a negative stain with
nigrosine and viewed at 1000x magnification
allowing the organism to be viewed in
with oil immersion, and C. Providencia rettgeri
grown on tryptic soy agar and prepared as a wet
their living state and to see the motility of
mount and viewed at 1000x magnification with
oil immersion.
each species. Because a cover slip was

placed over the smear, it was much more difficult to view each species at 1000x magnification

using oil immersion as the coverslip was prone to sliding around as the slide was being moved

because the coverslip was not fixed to the slide. Each species, when being compared to the

simple stain and negative stain techniques, seemed larger in size, especially P. rettgeri (Figure

3). It was not possible to see the individual links as previously seen for B. cereus, and was seen
as a continuous strand, like a worm. C. freundii was observed to have a darker staining head

with a clearer body (Figure 4). D. radiodurans was not very motile and had a bubble-like 3D

look to them when viewed at 1000x magnification under oil immersion (Figure 5). E. coli had

minimal motion with Brownian swimming as well as L. lactis. L. lactis was also very hard to

find under the microscope at 1000x.

Figure 4: The distinct darker head Figure 5: The three-dimensional

(A.) with a clearer body (B.) in
Citrobacter freundii which was
grown on a tryptic soy agar and
prepared as a wet mount and viewed
at 1000x magnification with oil
immersion.
bubble look of Deinococcus
radiodurans which was grown on a
tryptic soy agar and prepared as a
wet mount and viewed at 1000x
magnification with oil immersion.

The Gram stain technique had the largest margin of error. Preparation of two slides per

organisms was done in case of the event that a slide was not prepared correctly or the organisms

were not found on the slides. Organisms that resulted in a purple stain were Gram-positive

(indicating that the cell wall has a thick peptidoglycan layer) while organisms that were stained

pink were Gram-negative (indicating that the cell wall has a thick peptidoglycan layer). Table 2

below indicates whether the organisms were Gram-positive or Gram-negative:

Table 2: The Gram staining outcome of the six bacterial species (Bacillus cereus, Citrobacter
freundii, Deinococcus radiodurans, Escherichia coli, Lactococcus lactis and Providencia
rettgeri) which were grown in a tryptic soy broth and prepared using a Gram stain with crystal
violet, Grams iodine, ethanol, and safranin and viewed at 1000x magnification with oil
immersion.
Bacterial Species Gram Staining Outcome

Bacillus cereus Gram-positive

 Citrobacter freundii Gram-negative
Deinococcus radiodurans Gram-positive
Escherichia coli Gram-negative
Lactococcus lactis Gram-positive
Providencia rettgeri Gram-negative

C. freundii and P. rettgeri were unable to be found under the microscope because they

were undergrown as cultures (and therefore referral to the demonstration was necessary). B.

cereus was an older culture and therefore the stain did not hold as well. E. coli was very faint and

was more of a peach colour than a pink colour. C. freundii (on demonstration) again had a

distinct darker head and a clearer body as seen in the wet mount technique. D. radiodurans

had very distinct tetrads and were the easiest to find. L. lactis bacteria were dispersed and more

troublesome to find on the slide.

Finally, for determining the species within a mixture, the Gram staining experiment of

the known species was compared to the unknowns. The cell arrangement of the unknowns was

compared with the known species done in simple stains, negative stains, and wet mount

experiments. The combination of the Gram stain result and the cell arrangement helped

determine the unknown organisms within each mixture.

The mixture 1 broth contained both a Gram-positive species and a Gram-negative

species. The Gram-positive species had a diplococci arrangement while the Gram-negative

species had a staphylococci arrangement. In comparison to both L. lactis (Gram-positive and

diplococci) and E.coli (Gram-negative and staphylococci), it was concluded that both species

were the unknowns in the mixture (Figure 6).

 Figure 6: The comparison of the known bacterial species A. Lactococcus lactis which was
grown in a tryptic soy broth and prepared using a Gram stain with crystal violet, Grams
iodine, ethanol, and safranin and viewed at 1000x magnification with oil immersion and B.
Escherichia coli which was grown in a tryptic soy broth and prepared using a Gram stain with
crystal violet, Grams iodine, ethanol, and safranin and viewed at 1000x magnification with
oil immersion with C. the identified bacterial species within a mixture which was grown in a
tryptic soy broth and prepared using a Gram stain with crystal violet, Grams iodine, ethanol,
and safranin and viewed at 1000x magnification with oil immersion.

The mixture 2 broth also contained a Gram-positive species and a Gram-negative species.

The Gram-negative species was more numerous and easier to find than the Gram-positive

species (referred to the demonstration to find the Gram-positive species). The Gram-negative

species had a darker stained head from its body in a bacilli arrangement, like C. freundii. The

Gram-positive species had a tetrad arrangement like D. radiodurans. Concluding, the unknown

species of the mixture was C. freundii and D. radiodurans (Figure 7).

Figure 7: The comparison of the known bacterial species A. Citrobacter freundii which was
grown in a tryptic soy broth and prepared using a Gram stain with crystal violet, Grams
iodine, ethanol, and safranin and viewed at 1000x magnification with oil immersion and B.
Deinococcus radiodurans which was grown in a tryptic soy broth and prepared using a Gram
stain with crystal violet, Grams iodine, ethanol, and safranin and viewed at 1000x
magnification with oil immersion with C. the identified bacterial species within a mixture
which was grown in a tryptic soy broth and prepared using a Gram stain with crystal violet,
Grams iodine, ethanol, and safranin and viewed at 1000x magnification with oil immersion.
 Because the comparison the unknown species to the known species, it can be concluded

that it was possible to identify a species based on its cellular morphology. Below further

confirms the unknown species in both mixtures to the known species with Gram-staining

techniques:

Table 3: The cellular morphology and arrangement and the Gram staining outcome of the six
bacterial species (Bacillus cereus, Citrobacter freundii, Deinococcus radiodurans, Escherichia
coli, Lactococcus lactis and Providencia rettgeri) and the four unknown bacterial species in the
two mixtures (labeled Unknown 1 in Mixture 1, Unknown 2 in Mixture 1, Unknown 1 in
Mixture 2, and Unknown 2 in Mixture 2) which were grown in a tryptic soy broth and prepared
using a Gram stain with crystal violet, Grams iodine, ethanol, and safranin and viewed at 1000x
magnification with oil immersion. The unknown bacterial species were identified and compared
to the known bacterial species and are highlighted by colour.
Bacterial Species Cell Arrangement and Gram-positive or Gram-
Shape negative
Bacillus cereus streptobacilli Gram-positive
Citrobacter freundii single bacilli Gram-negative
Deinococcus radiodurans tetrads Gram-positive
Escherichia coli bacilli and diplobacilli Gram-negative
Lactococcus lactis staphylococci Gram-positive
Providencia rettgeri single bacilli Gram-negative
Unknown 1 in Mixture 1 staphylococci Gram-positive
Unknown 2 in Mixture 1 diplococci Gram-negative
Unknown 1 in Mixture 2 cocci; tetrads Gram-positive
Unknown 2 in Mixture 2 bacilli Gram-negative

Discussion

Are there any concerns for any of the performed experiments? There were very minimal

concerns for all experiments performed. Most arose from the wet mount and Gram staining

experiments. As mentioned previously, the wet mounts required a coverslip and therefore

viewing the organisms at 1000x magnification under oil immersion was difficult because the
coverslip was prone to sliding as the slide was being moved. As for the Gram staining

experiment, two preparations of each organism was done in case of the event that a slide was not

prepared correctly or the organisms were able to be found on the slides. C. freundii and P.

rettgeri in the Gram staining experiment were unable to be found on either preparation because

they were undergrown as cultures and therefore needed referral to the demonstration for

observation of the organisms. B. cereus was an older culture and therefore the stain for Gram

staining did not hold as well, which could have gave a false negative. Also for Gram staining, L.

lactis bacteria were dispersed and more troublesome to find on the slide. In determining the

unknown species for mixture 2, the Gram-positive species was not visible on the slide and

referral to the demonstration was required.

Is there confidence in the data? There were instances where referring to the

demonstration was required due to the fact that there were no results coming from the slides (for

example, bacterial species not showing on the slide). If the demonstration slides were not

available, there were other staining experiments to refer to when determining the unknown

species. Also, the mixture 2 of the unknown, D. radiodurans was not possible to see, therefore

determining an unknown that cannot be seen would have been impossible. As for the remaining

results for simple stain, negative stain, wet mount, and Gram stain experiments, results were

consistent for the morphology and arrangement of the organisms. Overall, the findings were

reasonable for the circumstances given.

Did any of the different techniques affect the conclusions? Any difference in techniques

were minor and helped to improve results. For example, keeping crystal violet stain on for two

minutes versus one minute for the simple stain experiment helped to increase the staining and

contrast of the cells.

 Was the hypothesis correct? Yes, by comparing known bacterial species to unknown

bacterial species, identification using cell morphology was possible. In comparing all the results

for one of the identified unknown organisms (for example, D. radiodurans), there was

consistency in the cell arrangement and morphology throughout all experiments (simple stain,

negative stain, wet mounts, and Gram stain)

Overall, there were small concerns about possible sources of error and outcomes each

experiment. The results were reasonable and there is confidence in the results as there was

consistency throughout the experiments. Small technical difference led to improved results. The

hypothesis was approved as the identification of the unknown bacterial species was successful.

Citations and References

1. McGreevy, A. Biology of Bacteria and Archaea: The Laboratory Manual. Winnipeg:

University of Winnipeg; 2017. p. 7 9.

2. McGreevy, A. Biology of Bacteria and Archaea: The Laboratory Manual. Winnipeg:

University of Winnipeg; 2017. p. 10 12.

3. McGreevy, A. Biology of Bacteria and Archaea: The Laboratory Manual. Winnipeg:

University of Winnipeg; 2017. p. 13 15.

4. McGreevy, A. Biology of Bacteria and Archaea: The Laboratory Manual. Winnipeg:

University of Winnipeg; 2017. p. 16.