Name : Joyleen Mutubuki

Reg no: H200638G

Program: Biotechnology

Course : Microbiology (SBT 120)

Lecturer : Mr T Chirova

Level : 1.2

Year : 2021

Title : Practical write up 2

Title : Using differential and selective media.

Abstract:
An experiment was conducted in which differential and selective media used where
selection can be achieved by altering the pH of the media.There were 6 bacteria that
were streaked onto MacConkey and EMB agar. The bacteria used were Pseudomonas
Aeruginosa, Escherichia Coli, Aspergillus Brasiliensis, Candida Albicans, Salmonella
and Bacillus subtilis.Some of the bacteria are gram negative and some ae gram
positive or lactose and non lactose fermenters.Tap water was streaked onto
MacConkey and Nutrient agar and no growth was observed. Lactobacillus was found
to be in lacto and a nose swab. In some of the petri dishes there were microorganisms
which were observed and some of the microorganisms were coloured and others were
shaped.

Introduction:

Bacteria it as a microorganism that contains prokaryotic cells with the cell walls that
contain the structural molecule peptidoglycan and they also have bacterial rRNA
(Prescott 2002).Bacterial is isolated or differentiated using selective and differential
media .Selective media is a media made up of specific components that promote the
growth of some bacteria and inhibit the growth of other bacteria.Selectivity is
achieved in three ways :1) by adding something to the media to discourage the

growth of species not required by altering the pH of medium.

2) by altering the pH of the medium .
3) by omission of some ingredients required by most bacteria

but not by the organism to be isolated.

For bacterial identification differential media are used.Differential media contain
specific biochemical indicators that demonstrate the presence of certain substances
characteristic of certain bacteria.These differential media they may contain certain
dyes and chemicals that will result in specific growth patterns that allows specific
bacteria to be identified ( Tortora et al 2013).

MacConkey agar is both a selective and differential media used to differentiate

between members of the Enterobacteriaceae family ,since it contains lactose ,and
neutral red dye ,bacteria that catabolise lactose by fermenting it release acidic waste
products that make colonies appear pink to red in colour (Prescott 2002).The red
colour is due to the production of acid from the lactose, which causes the pH to drop
and when the pH of media drops below 6.8 the dye becomes red. Non-lactose
fermenting bacteria are colourless and transparent and do not change the appearance
of the media.MacConkey agar is used for the detection of E .Coli and related bacteria
in water supplies and elsewhere.This media contain dye that suppress the growth of
gram positive bacteria .MacConkey agar it also contain bile salts ,bile salts favour the
growth of gram negative bacteria by inhibiting the growth of gram positive
bacteria( Prescott 2002).

EMB ( Erosin methyl blue ) agar is both a selective and differential media which is
made up of two dyes ,erosin Y and methylene blue .It inhibit the growth of gram
positive bacteria and it promotes the growth of gram negative bacteria .It differentiate
bacteria based on if they are lactose fermenters or not .Lactose fermenters produce
coloured colonies of either green or purple example fecal bacteria like E.Coli .Non
fermenters produce colourless colonies. Nutrient agar( NA) is a general purpose
nutrient media. It is neither differential nor selective. It contains many nutrients
needed by bacteria and fungi in order for them to grow.It promotes the growth of non
fastidous microorganisms ( Tankenshwar 2016).

Motility agar it is a colourless salt that helps determine bacterial motility .This dye
gets incorparated into cells and gets reduced to a red pigment and this red pigment
shows where the bacteria grows .Motility agar works by using tetrazolium salt to stain
the bacteria .If the stain which is red only shows on the stabline of the agar it means
that the species is not motile .If the bacteria is motile it will move away from the line.
Salmonella is a gram negative bacteria. Most strains of Salmonella are nonlactose
fermenters on MacConkey agar, although there is a very small number (1%) that are
able to ferment lactose (McDonough et al, 2000).

Bacillus subtilis is a genus of gram positive bacteria ,rod shaped bacteria,gram

positive bacteria that does not ferment lactose (Ramos et al, 2000).Candida Albicans
is a gram positive fungi and it does not ferment lactose (Gow and Yadav, 2017).

Escherichia Coli is a gram negative, rod shaped bacteria (Singleton, 1999). They are
also lactose fermenting and form pink colonies when cultured on MacConkey Agar.
Aspergillus Brasiliensis produces colonies that are white or yellow in colour but over
time darken to a brown colour (Varga et al, 2007).Pseudomonas Aeruginosa is a gram
negative bacteria that is not a lactose fermenter (Vasil, M. L., 1986).

When all bacteria like E .Coli are streaked into different selective and differential
media their mophological characteristised have to be observed different types of
shapes that the bacteria maybe spherical (coccus) ,rodshaped ( Bacillus subtilis ).So
asceptic techniques like auto claving ,passing the container with the bacteria over the
flame so as to reduce the contamination of cultures .When the petri dishes are being
streaked the lid should not be fully opened because some of the microorganism in the
air might contaminate the media hence leading to the unexpected results. Safety
precautions should be taken when streaking bacteria like salmonella because it cause
gastrointestinal illness and fever called salmonellosis.So it is wise to wash hands or to
disinfect the work area with 70% alcohol so as to reduce cntamination.

Aims and objectives:

Aim: to learn how to differentiate bacteria based on its morphology

Objectives: -to observe the growth of bacteria under selective and differential media.
-to determine the number and type of organism that can grow on different
media.

Methodology:
Materials:
- 3 plates of MacConkey and EMB media
-5 know broth cultures
-1 uknown broth cultures
-6 tubes of Hugh and Leifson’s lactose medium
-6 tubes of motility medium
-NA
-MRS agar
-Tap water
-lacto
-Sterile swabs
-glass spreader

Method:

Growth of microoroganism on complex media:

1. 3 MacConkey and 3 EMB plates were divided into half and innoculated them with
6 bacterial species provided.
2. The 6 plates were then incubated at 37℃ for 48 hours .
3. The various cultures on the pplates were observed and described.
4. A table of results summarizing the growth and properties of all bacteria on the 2
media was then generated.
Effect of type and media on growth of microorganism:

Tap water:

1.0,1 ml of tap water wass pippetted on each of the plate provided( NA ,MacConkey).
2. A glass spreader wa dipped into 70% ethanol and allowed to cool .
3. A sterile spreader was used to spread the water evenly on the surface of each plate.
4. The plates were inverted and incubated at 30℃ for 24 -48 hours.

Lacto:
1. A loopful of lacto wa streaked on each of the plate provided (NA ,MRS,
MacConkey).
2. The plates were incubated at 30℃ for 24-48 hours .

Nose swab:
1. The interior of the nose wa rubbed using the two sterile swabs which were
provided.
2. One of the swabs was rubbed over one quater of NA agar plate .
3. The step 2 was repeated and the other swab on EMB agar.
4. All plates were incubated at 30℃ for 24- 48 hours.
5. Obervations were recorded and tabulated.

Results:
 Some of the bacteria which were streaked in different selective and differentail
media managed to grow and in some petri dishes there were no observable
changes .Some of the bacteria were pink and some white.

Table 1 :Growth of microorganism on MacConkey and EMB

Bacteria MacConkey Agar EMB Agar

Pseudomonas Aeruginosa No observable change No observable change
Escherichia Coli No obseravble change. No growth
White fungal
contamination
Aspergillus Brasiliensis No observable change No growth
Green fungal
contamination
Candida Albicans Growth observed No observable change.
Pink, round and flat
Salmonella Growth observed No growth
Pink, round and raised White fungal
contamination
Bacillus subtilis No observable change No growth
White fungal
contamination
Table 2 : Effect of media on growth of microorganism

MacConkey Agar Nutient Agar MRS Agar

Tap water No observable No growth No observable
change. Contamnation of change.
white fungal growth
And 2 cream round
colonies ( one small
and one big)
Lacto Growth observed Growth observed Growth observed
on streaking line Small, cream, round Small,cream, round
and raised and raised
 Small, cream,
round and raised
Nose Swab No observable Growth observed Growth observed
change. Many white, rasied Few white, raised
and round colonies colonies

Discussion:

Since Pseadomonas Aeroginosa is a gram negative bacteria .Bacterial growth was

expected in MacConkey agar since MacConkey agar is aselective media for gram
negative microorganism (Prescott 2002).The was no bacterial growth in MacConkey
or EMB this was because there was overful in the incubator this might have affected
the incubation therefore preventig the growth of the bacteria.Since E .Coli is a gram
negative bacterial it was expected to observe microorganism growth on MacConkey
because MacConkey is used for the detection of E coli and other related bacteria in
water supplies ( Prescott 2002) therfore there was no growth this might be due to the
bacteria which was collected in death phase.

For Samonella there was no growth on EMB but there was growth of pink, round and
raised colonies on MacConkey Agar. The growth on MacConkey agar was expected
because MacConkey is a selective media for gram negative microorganisms and
Salmonella is a gram negative. Some strains of Salmonella are lactose fermenting
which would explain the pink colour of the colonies. The colonies become pink when
the microorganism is lactose fermenting (McDonough et al, 2000).Candida Albicans
had growth of pink, round colonies on MacConkey and no growth on EMB. The
result in MacConkey was unexpected. Candida Albicans is gram negative and not
lactose fermenting(Gow and Yadav, 2017) and sothere should not grow nor should
the colonies be coloured. These unexpected results could be as a result of mislabelling
of the bacteria. There could also have been cross contamination that occurred during
the streaking process.

Bacillus Subtilis had no growth on both MacConkey and EMB. This result is expected
because Bacillus Subtilis is gram positive and MacConkey is selective for gram
negative microorganisms ( Prescott 2002). The fungal contamination that was present
in the petri dished was possibly due to contamination from the surrounding area. This
indicates that the agar was not poured into the petri dishes aseptically. The fact that
similar contamination was in multiple petri dishes indicates that the contamination
occurred before the microorganisms were introduced onto the agar.

Aspergillus Brasiliensis had no growth on both MacConkey and EMB.This

observation was expected since Pseudomonas Aeruginosa is a gram negative bacteria
that is not a lactose fermenter (Vasil, M. L., 1986) where as MacConkey agar is a
differential media which differentiate between lactose fermenting and non lactose
fermenting gram negative bacteria.There was no growth in MacConkey agar with tap
water. This indicates that there was no gram negative microorganisms present in tap
water. There was also no growth in Nutrient agar. Since Nutrient agar is not selective
it indicates that there were no microorganisms present in the tap water. The
contamination could have been due to aseptic techniques not being carried out
correctly.
With the lacto there was growth observed on MacConkey agar, Nutrient agar and
MRS agar. This indicates that the bacteria in the lacto was gram negative as
MacConkey is selective for gram negative bacteria. MRS is selective for
Lactobacillus. The growth that was observed indicates that there was Lactobacillus
present in the lacto. The growth on Nutrient agar was expected as it possesses
nutrients needed for most bacteria to grow. There was growth observed on both
Nutrient agar and MRS agar for the nose swab. There was growth and this was
expected on Nutrient agar as the nose usually possesses healthy bacteria and Nutrient
agar allows all bacteria to grow. The growth on MRS indicates some of the bacteria
was Lactobacillus. MRS had far fewer colonies than Nutrient agar. This indicates that
there was more than one type of bacteria present in the nose. The MRS is selective
and only allows certain bacteria to grow while Nutrient agar allows a lot of bacteria to
grow on it.

Conclusion:

Since the aim of the practiacal was to learn how to differentiate bacteria based on its
morphology and the objectives of the practical wereto observe the growth of bacteria
under selective and differential media ,to determine the number and type of organism
that can grow on different media. The results in table 1 and 2 where there was no
growth and where there were growth in some of the media this shows that the
practiacl was succesful since the aim and objectives of the practiacl were managed to
be achieved .
Reference:
th
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3Varga. J., Kocsube. S., Toth. B., Frisvad. J. C., Perrone. G., Susca. A., Meijer. M.
and Samson. R. A., 2007, Apergillus Brasiliensis, a biseriate black Aspergillus
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