A sample lab report

Topic:_________________________________

Introduction
The study of microbiology requires not only an academic understanding of the
microscopic world but also a practical understanding of lab techniques and
procedures used to identify, control, and manipulate microorganisms. The
proper identification of a microorganism is not only important in a microbiology
lab but also in the medical, industrial, and pharmaceutical fields. In this lab
report, lab techniques and procedures learned during this course were
performed to assess each students’ practical knowledge in microbiology.

The goal of this lab report is 1) to demonstrate comprehension of the methods

and lab techniques learned during the semester 2) to explain the tests
performed on each isolated unknown that led to the identification of each
unknown 3) and to give a background on the characteristics, pathogenicity and
some uses of one of the identified unknowns.

Materials and Methods

At the beginning of this project, the professor handed each student a numbered
test tube containing two unknown bacteria (one Gram positive and one Gram
negative). The test tube used in this study was tube 109. The instructor asked
that each student isolate the two unknown bacteria and then identify each. The
lab techniques and procedures used throughout this experiment were from
McDonald’s laboratory manual (4).

To start, the two unknown bacteria had to be successfully isolated. Using a wire
loop, a small sample of the numbered test tube was plated on a nutrient agar
using the quadrant streak method (4). The numbered test tube was then placed
in a refrigerator to slow down any continued growth. The nutrient agar was
labeled “Isolation 1” and was placed in an incubator for 48 hours at 37 degrees
Celsius. After 48 hours, the Isolation 1 plate was observed. There were 34
isolated colonies present, all of which showed the same growth pattern. Four
were chosen at random and Gram stains were performed on each (procedure
for the Gram stain was used according to McDonald’s laboratory manual). Each
of the four Gram stains showed positive rods. Seeing that no negative Gram
stains were observed, a different approach was taken. Instead, a small sample
of the numbered test tube was streaked using the quadrant isolation method on
an EMB (eosin-methylene blue) plate and then on an MSA (mannitol salt agar)
plate (4). The EMB plate inhibits Gram positive bacteria and the MSA plate
inhibits gram negative bacteria (4). So, growth on each plate should favor only
one of the unknowns. The EMB plate was labeled “EMB 1” and the MSA plate
was labeled “MSA 1.” Each was placed in the incubator for 48 hours at 37
degrees Celsius. After 48 hours, each plate was observed. EMB 1 had very little
growth so it was returned to the incubator and allowed to grow for another 48
hours. MSA 1 had significant overgrowth and was discarded. A new MSA plate,
labeled MSA 2, was created using the same quadrant isolation streak method. It
was only let to incubate for 24 hours at 37 degrees Celsius instead of 48 hours.
After 24 hours, MSA 2 was observed. It had many well isolated colonies. One of
the colonies was transferred to another MSA plate, labeled MSA 3, using the
quadrant isolation streak. (This was performed to ensure that the Gram positive
bacteria were well isolated from the Gram negative.) MSA 3 was placed in the
incubator at 37 degrees Celsius for 24 hours. After another 24 hours, both MSA
3 and EMB 1 were observed. Both EMB 1 and MSA 3 had well isolated colonies.
One of the colonies from the MSA 3 plate was Gram stained. It showed Gram
positive rods. A loopful of bacteria from that same colony from MSA 3 was
transferred to a nutrient agar plate labeled “NA positive stock.” This nutrient
agar plate became the stock plate for Gram positive bacteria for the rest of the
experiment. One of the well isolated colonies on the EMB 1 plate was
transferred to a new EMB plate, labeled EMB 2, using the quadrant isolation
streak method. (This was to ensure that the Gram negative bacteria were well
isolated from the Gram positive on the plate.) Both the EMB 2 plate and the NA
positive stock plate were placed in the incubator at 37 degrees Celsius. After 72
hours, one of the well isolated colonies on EMB 2 was Gram stained. Gram
negative rods were observed. A loopful of bacteria from that same colony was
transferred to a new nutrient agar plate and labeled “NA negative stock.” It was
placed in the incubator at 37 degrees Celsius. At this point, both bacteria were
successfully isolated and plated on stock nutrient agar plates.

Various tests were conducted on each the unknown bacterial cultures. An

explanation of each test and the results are in Table 1 (for the Gram positive
bacteria) and Table 2 (for the Gram negative bacteria). The tests were
performed in such a way that each test eliminated a possible unknown
candidate. Chart 1 details the sequence of tests performed on the Gram positive
bacteria and Chart 2 details the sequence of test performed on the Gram
negative bacteria.
 

For the gram positive bacteria, the following tests were performed:

1)      Urea

2)      Catalase

3)      Casein

4)      Methyl Red

5)      Glycerol

6)      Simmons Citrate

For the gram negative bacteria, the following tests were performed:

1)      Nitrate

2)      Simmons Citrate

3)      Urea

4)      Casein

5)      Voges-Proskauer (V-P)

6)      Sorbitol

*All of the tests were performed and explained as described in the McDonald
Laboratory Manual
 
Results
 
Table 1. Results of Gram Positive Unknown

REAGENTS or
TEST OBSERVATIONS RESULTS INTERPRETATI
MEDIA
Crystal violet,
Gram positive
Gram Stain Gram’s Iodine, Purple Rods
rods
alcohol, safranin
Urea Urea tube No color change; Negative The Gram pos
remained yellow bacteria is una
produce ureas
hydrolyzes ure
carbon dioxide
 ammonia
The Gram pos
bacteria posse
Hydrogen enzyme catala
Catalase Bubbling Positive
peroxide breaks down h
peroxide into o
gas and water
The Gram pos
bacteria is una
No clearing around
Casein Milk Agar Negative produce casea
bacteria
degrades the c
protein in milk
After adding methyl red
to test tube, color The Gram pos
MR-VP tubes, changed from light bacteria does
Methyl Red Negative
methyl red dye yellow to a darker produce acid d
yellow; no red was glucose catabo
present
After inoculation and The Gram pos
Glycerol Glycerol tube incubation, the tube Negative bacteria does
remained red ferment glycer
The Gram pos
Simmons Citrate Agar changed color bacteria produ
Citrate Positive
agar from green to blue enzyme citrase
breaks down c
 

Table 2. Results for Gram Negative Unknown

REAGENTS or
TEST OBSERVATIONS RESULTS INTERPRETATIONS
MEDIA
Crystal
violet,
Gram
Gram’s
Gram Stain Pink Rods negative
iodine,
rods
alcohol,
safranin
Nitrate broth
tubes, nitrate After addition of
Gram negative
reagent A, reagents A and
Nitrate Positive bacteria can reduce
nitrate B, color was deep
nitrate to nitrite
reagent B, red
powered zinc
 The Gram negative
Agar changed bacteria produces
Simmons
Citrate color from green Positive the enzyme citrase,
Citrate agar
to blue which breaks down
citrate
The Gram negative
bacteria is unable to
produce urease,
No color change;
Urea Urea tube Negative which hydrolyzes
remained yellow
urea to carbon
dioxide and
ammonia
The Gram negative
Clearing around
Casein Milk Agar Positive bacteria is able to
bacterial streak
produce casease
The Gram negative
MR-VP tubes, After addition of
Voges- bacteria does not
VP reagent reagents A and
Proskauer Negative produce acetoin as
A, VP B, the tube
(VP) an end product of
reagent B turned yellow
glucose fermentation
Color change Gram negative
Sorbitol Sorbitol tube from red to Positive bacteria ferments
yellow sorbitol
 
 

Discussion/Conclusion
The Gram positive unknown was identified to be Bacillus subtilis. The instructor
verified this to be correct. This deduction was reached with several bits of
evidence. Firstly, the Gram positive unknown was rod shape. This narrowed it
down to both of the Bacillus species. However, the researcher felt it was
important to perform a battery of tests in order to confidently conclude that the
Gram positive unknown was indeed B. subtilis. So, the pre-constructed
sequence of tests was still performed as if all five of the Gram positive options
were still possible. Secondly, the series of tests performed on the Gram positive
unknown led to B. subtilis as the only possible candidate. A negative urea test
removed S. epidermidis as a possibility. A positive catalase test removed E.
faecalis as a possibility. A negative casein test removed S. aureus as a
possibility. And, a negative methyl red test removed B. cereus as a possibility.
This left only B. subtilis. The citrate test and the glycerol test were both done to
assure accuracy of the identification of B. subtilis as the Gram positive
unknown. The citrate test was positive and the glycerol test was negative. Both
of these tests concurred with the expected results of B. subtilis metabolism.
The Gram negative unknown was identified to be Pseudomonas aeruginosa. The
instructor verified this to be correct. This deduction was reached for a couple
reasons. Firstly, each of the tests in the series ruled out a possible candidate
until P. aeruginosa was the only possibility left. A negative nitrate test ruled
out E. coli. A positive citrate test ruled out P. vulgaris (and reinforced the
decision that E. coli was not the unknown). A negative Urea test removed K.
pneumoniae (and reinforced the decision that P. vulgaris was not the unknown).
A negative casein test ruled out E. aerogenes as a possibility. This left only P.
aeruginosa as the unknown. A VP test and a sorbitol test were performed to
validate that P. aeruginosa was the unknown. A negative VP test and a positive
sorbitol test concurred with the expected results of P. aeruginosa metabolism.
No difficult problems occurred with the identification of B. subtilis. The only
slight issue was that B. subtilis grew extremely quickly and overgrowth was a
common problem. For example, a nutrient agar stock plate for B. subtilis was
inoculated almost daily to ensure the cultures used for each of the identification
tests were young.
The Gram negative unknown, however, was very difficult to identify. Firstly, all
of the Gram negative unknown possibilities were rods. This rendered the Gram
stain useless in identifying P. aeruginosa based on shape. Secondly, the culture
was very difficult to isolate because it grew extremely slowly on the EMB plates
and a pure culture took two weeks to achieve. Thirdly, several of the tests had
to be repeated because the first stock plate of P. aeruginosa was found to be
contaminated. All of the tests had to be thrown away and repeated using a
newly grown stock plate of P. aeruginosa. After a second round of tests, all of
the results matched up with the expected results of P. aeruginosa except for the
VP test. It turned up positive. It was performed for a third time and the result
was negative. The second VP test was probably contaminated and was not a
reliable test result.
 

Background Information on Bacillus subtilis

Bacillus subtilis is a very well-studied microbe. It is considered the “type”
species of the Bacillus genus (2). B. subtilis, of the Family Bacillaceae, was
named in 1872 by Ferdinand Cohn (3). This bacterium is also known by the
names hay bacillus or grass bacillus (1). Like other bacteria in
the Bacillaceae family, B. subtilis can grow in the presence of oxygen (with the
help of its catalase enzyme) and is endospore-forming (1). It is predicted that it
spends most of its time inactive and in spore form. B. subtilis is commonly
recovered from soil, water, air, and decomposing plant material (1). Different
strains of B. subtilis can be used as biological control agents under
differentsituations. One of the B. subtilis strains produces a chemical called
iturin (1). This chemical acts as a growth inhibitor for many other bacteria and
some fungi, which allows B. subtilis to out-compete several other soil-living
microorganisms (1).  Because of this strains’ inhibitory affect on many other
bacteria, it is sometimes added to fertilizer to help delicate plants to grow (1).
Furthermore, some probiotic companies are using B. subtilis in their probiotics
because of their inhibitory affect on many pathogenic enteric bacteria (2). Some
research, however, questions whether it is wise to put an endospore-producing
bacteria in a human’s gut even if it currently is not pathogenic to humans.
Contrary to this, studies have found that B. subtilis sometimes naturally inhabits
the intestinal tracts of humans without causing any harm (2). As it currently
stands, B. subtilis is believed not to be pathogenic to humans. Some other
commercial applications of B. subtilis include: cleaning agents in detergents,
leather processing, preparing special Japanese and Korean dishes, starch
modification, and several other specialized chemicals (3).
 

References
1. “Bacillus subtilis.” Organic Resource Guide: Material Fact Sheet.
http://www.cefs.ncsu.edu/newsevents/events/2010/sosa2010/20101013tomato/pro
duct01-bacillussubtilis.pdf. Accessed April 27, 2014.
2. Cartwright, Peter. “Bacillus subtilis: Identification & Safety.” Protexin: 2009.
http://www.protexin.com/attachments/Probiotic%20News%20Issue%201.pdf.
Accessed April 27, 2014.
3. O’Keeffe, Jillian. Environmental and Industrial Use of Bacillus Subtilis. Global
Post. http://everydaylife.globalpost.com/environmental-industrial-use-bacillus-
subtilis-30152.html. Accessed April 27, 2014.
4. McDonald, Virginia et al. Lab Manual for General Microbiology. Saint Louis
Community College at Meramec, 2011.