Purpose:

To learn how to use Bergey’s Manual and to learn what are commonly used some media used in growth dependent identification methods such as BA, EMB, MC, SS, XLD, TSIA, BSA, DNase Test Agar, HE, MSA which are told in our manual.
To perform three types of growth dependent identification methods such as use of BA, EMB, SS agar
To be familiar with nutrient slant culture, nutrient broth culture, nutrient gelatin stab culture, nutrient agar plate culture.
To learn and be able to distinguish between selective media and differential media.
To be familiar with the types of differential and selective media and for identification of which microorganisms they are used.

Procedure:

In general:

Use aseptic techniques and perform the inoculations for nutrient slant culture, nutrient broth culture, nutrient gelatin stab culture and nutrient agar plate culture.
Incubate tubes and evaluate the results he following week
Each group performs inoculations onto nutrient agar, salmonella shigella agar, eosin methylene blue agar and blood agar each of them using different bacteria.
The following week observe and evaluate the results.

Procedures in separate parts

Under aseptic conditions take given bacterial culture –for our group Staphylococcus aureus- with inoculating needle then followings…

Nutrient Agar Slant Culture: Inoculate the bacteria on the agar slant by drawing the needle upwards in a straight line starting from the bottom of the slant. Incubate the agar slant at 37°C for 48 hr. Next week observe the colony characteristics according to the manual.

Nutrient Broth Culture: Inoculate the bacteria in the NB with inoculating loop. Incubate the agar slant at 37°C for 48 hr. Next week observe the colony characteristics according to the manual. Take care not to agitate the tube.

Nutrient Gelatin Stab Culture: Inoculate the bacteria into the gelatin stab with inoculating loop by inserting the needle to the ¾ of the stab. Incubate the agar slant at 20°C for 1 week. Next week observe the colony characteristics according to the manual.

Nutrient Agar Plate Culture: Inoculate the bacteria on the NA plate with inoculating loop by streak plate technique. Incubate the agar slant at 37°C for 48 hr. Next week observe the colony characteristics according to the manual.

Eosin Methylene Blue Agar (EMB): Inoculate the bacteria on the EMB Agar with inoculating loop by streak plate technique. Incubate the agar slant at 37°C for 24 hr. Next week observe the colony characteristics according to the manual.

Blood Agar (BA): Inoculate the bacteria on the Blood Agar with inoculating loop by streak plate technique. Incubate the agar slant at 37°C for 24 hr. Next week observe the colony characteristics according to the manual.

Salmonella Shigella Agar (SS): Inoculate the bacteria on the SS Agar with inoculating loop by streak plate technique. Incubate the agar slant at 37°C for 24 hr. Next week observe the colony characteristics according to the manual.

 

Results:

Group
Bacteria species
NA
SS
EMB
Blood Agar
Gelatinase activity
1
Staphylococcus aureus
Whitish
Brownish
Pink
No hemolysis (gamma)
–
2
Enterobacter aerogenes
Whitish
Colourless
Pink
Alpha-hemolysis
–
3
Escherichia coli
Whitish
Light brown (orange-yellow)
Pink
Beta-hemolysis
–
4
Salmonella anatum
Whitish
Black dots
Pink
Alpha-hemolysis
–
5
Proteus vulgaris
Whitish
brownish
pink
Alpha-hemolysis
–
6
Bacillus thuringiensis
Whitish
No growth
No growth
Beta-hemolysis
–
7
Serratia marcescens
Pinkish
Pinkish
Pink-purple
Alpha-hemolysis
+
8
Bacillus subtilis
Brownish
No growth
No growth
Alpha-hemolysis
–
9
Salmonella anatum
Whitish
Black dots (light brown)
Pink
Alpha-hemolysis
–
Expected results based on Bergey’s manual:

Group
Bacteria species
NA
SS
EMB
Blood agar
Gelatinase activity
1
Staphylococcus aureus
Circular, Orange to white
No growth
No H2S
No growth
Beta-hemolysis
(+)Saccate liquefaction with white yellowish sediment
2
Enterobacter aerogenes
Rough
No pigment
Pinkish red, cream colored
Pink colonies with no metallic green shine
α hemolytic
Differs among strains (11-80% positive)
3
Escherichia coli
White-yellowish,
moist glistering, spreading growth
Pinkish red,
No H2S
Metallic green sheen,
Blue black colonies

Some being hemolytic
(-)Grayish white, no liquefaction
4
Salmonella anatum
White-red,
Grayish, smooth, moist
Colourless colonies with black centres,
H2S production

Slow lactose utilization, pink
γ hemolytic
(-)Flat, surface growth, no liquefaction
5
Proteus vulgaris
Opaque grey,
Bluish gray
Colourless, usually with black center
No lactose utilization, pink large colonies
γ hemolytic
(+) Rapid stratiform, liquefaction
6
Bacillus thuringiensis
White
No growth
No growth
β hemolytic
(-), No Growth
7
Serratia marcescens
White-red, pink, Round
colorless colonies
purple colonies
α hemolytic
(+), Infundible-form
8
Bacillus subtilis
White
No growth
No growth
γ hemolytic
(+) Liquefaction crateriform, to stratiform
9
Salmonella anatum
White-red,
Grayish, smooth, moist
Colourless colonies with black centres,
H2S production

Slow lactose utilization, pink
γ hemolytic
(-) Flat, surface growth, no liquefaction

Discussion:

In this experiment, we have learned several basic molecular biology techniques and use of equipments such as use of Bergey’s Manual to compare experimental results and theoretical results and types of media used in growth dependent identification methods of bacteria.

For our experiment we used three types of growth dependent identification methods and we used salmonella shigella agar, eosin methylene blue agar, blood agar as selective and differential media. Most of the results are expected but there are also some unexpected results which most probably caused by some errors during observation of color or it might be due to strain of bacteria which used. Moreover, we have lost samples of the NA slants and NB cultures. Therefore we couldn’t observe the NA slants and NB cultures. We only could observe the nutrient gelatin stabs, NA, EMB agar, Blood agar, SS agar plates.

In EMB agar that is both a selective and differential medium we use to differentiate Enterobacteriaceae species. EMB selects for negative bacteria and differentiates between lactose ferment from not ferment. EMB plates inhibit the growth of gram positive bacteria. Depending on the acid production, if a very small amount of acid is produced a pink color is observed while if a large amount of acid is produced a metallic greenish shine is observed. If there is not any fermentation of lactose is present, culture is colorless. In our group we observed pink colored colonies of S. aureus which is a gram positive bacteria, this means that it has low amount of lactose usage although we expected not to see any growth. I think this result is due to strain of bacteria; it might have somehow resistant to selection by eosin methylene blue.

Blood agar is a differential medium depending on the hemolysis which causes the lysis of red blood cells. There are especially three degrees of hemolysis. First of all, Beta hemolysis is a complete hydrolysis of RBC and hemoglobin. On the plate it seems like complete clearing around colonies. Secondly, Alpha hemolysis is a partial hemolysis and results in greenish discolorization around colonies. Finally, No hemolysis, which is also called as gamma hemolysis, results in no change in medium. In our group we observed no activity of hemolysis produced by S. aureus, this means that it does not have production of enzyme although we expected to see complete hyrolysis- beta hemolysis-. I think this result is again due to strain of bacteria, it might have somehow lost its ability to produce related enzyme. It is also possible that we can conlude by looking EMB agar test and blood agar test that it is not S. aureus (?!).

Finally, we used Salmonella Shigella (SS) Agar as a selective media for Slamonella and Shigella species. Culture includes chemicals such as bile salts, sodium thiosulfate and citrate which inhibit growth of all gram positive bacteria and most of gram negative ones. Moreover, the presence of red colonies reflects the lactose-fermenting species. According to Bergey’s Manual we expected to see No growth, and No H2S production of our species S. aureus. However, again we observed brownish colony formation. This also shows that it is not S. aureus or we always used wrong media for our group.

When we come to results of Nutrient gelatin stab culture, we can say that when bacteria did not procedure gelatinase, we expected that gelatin will sill stay intact. If the bacteria can produce gelatinase, then the gelatin will stay liquid that is a positive result. But, some bacteria also produce a little amount of enzyme. Therefore, in such cases, we have to incubate the media for up to 2 weeks. After two weeks if the results are still negative it means bacteria can not produce gelatinase. When we consider S. aureus, result is again opposite of expected. There is really a big problem with our species or strain.

Note that nutrient agar inoculation was as a control and used to see usual properties of colony formation of species and they were resulted as expected.

It is expected from us that only we discuss results of our group but I want to simply talk about other species in general.

Result for Enterobacter aerogenes was as expected but we have a problem at SS culture that we could not to see a pinkish color, we observed colorless. Could we explain it by death during inoculation?

Result for E.coli was not as expected but we have a problem at SS culture that we observed brownish dots showing H2S production. Moreover, in EMB we observed a pink color but we expected a metallic green shine. Could we explain it by a possible contamination or a difference of expression?

Result for Salmonella anatum was as expected and similar in two groups but we have a problem at Blood Agar that we observed α Hemolysis although expected is gamma (no) hemolysis.

Result for Proteus vulgaris was not as expected we only observed SS and EMB cultures as expected.

Result for Bacillus thrungiensis was as expected for both NA and BA media but we have a problem at SS and EMB agars. Could we explain it by a possible contamination? Moreover, for gelatinase activity we expect to see an activity but we did not. Could we explain it by low level of expression of enzyme gelatinase?

Result for Serratia marescens was as expected and best one when we compared the others. We almost observed just the expected results as written in the Bergey’s Manual.

Result for Bacillus subtilis was mostly as expected but we have a problem at Blood agar showing alpha hemolysis which is unexpected from a gram positive bacterium. We expected to see gamma hemolysis.

In conclusion, these exercises were successful and they were a good and essential part of study and learn microbiology. New procedures were practiced, and further understanding of difference between selective media which contain –generally- antibiotics for selection and differential media which contains indicators were gained. However, some media have properties of two of them, i.e., they have both selective and differential properties EMB Agar.

REFERENCES:

Picture: Retrieved November 24, 2005 from
http://textbookofbacteriology.net/staph.html.

Infromation: Retrieved November 24, 2005 from http://www.mc.maricopa.edu/~johnson/labtools/Dbiochem/emb.html

Bergey’s Manual of Determinative Bacteriology, 9 edition. Retrieved November 23, 2005 fromhttp://grove.ufl.edu/~jbjones/Pseudomonas_2004.pdf

John G. Holt [et. al.]. Bergey’s Manual® of Determinative Bacteriology. 9th Edition, 1994,The Williams Wilkins Company, Baltimore

Robert S. Breed, E. G. D. Murray, Nathan R. Smith. Bergey’s Manual® of Determinative Bacteriology. 7th Edition, 1957,The Williams Wilkins Company, Baltimore