Microbial Biometric Analysis Of Cells Found In Soil

Vusimuzi Percival Nxumalo (216035131)

Discipline of Microbiology

School of Life Sciences

University of KwaZulu-Natal

21 April 2017

Abstract

Microbial biometric or phenotypic analysis is the identification of microbial cells, by

isolation and microscopically, particularly those are found in soil. The microbial cells
examined was genus Bacillus, the unknown isolate was identified to be Bacillus cereus
species. This species was identified to be Gram-positive, when viewed under light
microscopy, and endospore forming isolate which give them advantage of surviving
harsh conditions. Bacillus cereus is the most common contaminant found in soil,
insects, and in mammalian intestines and gut where it competes with other intestinal
dwellings which are pathogenic, microbes like Salmonella that causes blood borne
diseases era eradicated by the presence of B.cereus, it is therefore advantageous but
only in acceptable amounts. The importance of this species in industries have brought
great momentum all over the world due to its wide range of enzyme secretion.

Introduction

Microbial diversity is an unseen international resource that deserves a greater

attention (Saikia et al.,2011). The are many reasons why knowing how to perform
some microbial identification. There are techniques used to identify phenotypes of
microbes. Phenotypic identification is the analysis of microbial species. There are
various types of microorganisms found almost everywhere, especially in the soil where
they are able to reciprocate in different ways with their symbionts. Microorganisms
includes genus Bacillus with its significant various activities and roles it plays. Bacillus
is the member of the phylum Firmicute which is Gram positive with rod-shaped cells
and can be obligate aerobes (oxygen reliant), and or facultative anaerobes (both
aerobic and anaerobic), and have the diamino acid which is meso-diaminopimelic acid
(meso-DAP direct) (Bergeys manual of firmicutes Schleifer and Kandler 1972).

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There are many species of the genus Bacillus, among those is the Bacillus cereus,
B.cereus is an aerobic, motile, beta hemolytic bacterium commonly found in soil and
food. Some strains of B.cereus are harmful (virulent) while others are advantageous
serving as probiotic for animals and are facultative anaerobes. Bacillus cereus
group, like all the members of Bacillus, can produce protective endospores, which
are dormant and non-reproductive structures that can survive harsh environmental
conditions. Endospore formation is usually triggered by lack of nutrients and usually
occurs in Gram-positive bacteria species. It was from this species that two new
enzymes, named AlkC and AlkD, which are involved in DNA repair, were discovered
in 2006 (Alseth et.,al 2006).

In the 2010s examination of the warning letters issued by the US Food and Drug
Administration issued to pharmaceutical manufacturing facilities addressing facility
microbial contamination revealed that the most common contaminant was B.cereus
species (Sandle et.,al 2014). At 10o C to 50o C this species tends to have maximal
growth, this is its optimal temperate range, one would conclude that they are
mesophilic. Despite of the wide diversity of the genus, most Bacillus species will
grow well on routine media such as nutrient agar but mostly, likely grow well on
blood agar. Bacillus cereus species can be regarded as parasitic microorganisms
since they utilize their host cells to obtain nutrients, hosts like human large intestines
and insects etc. It is found in humans when they have ingested food contaminated
by B.cereus, having the ability of living in wide range of habitats due to its potential to
metabolize a range compounds such as proteins, amino acids, carbohydrates and
peptides from the surrounding environment. Metabolism of the above mentioned
compounds indicates the presence of enzymes, protease, amylase and other
enzymes that enables it to survive.

The utilization of organic chemicals tells us that it is a chemoorganotroph, however it

is also heterotrophic since it obtains carbon from other organic sources rather than
CO2. With all characteristics and functions that B.cereus has, ecologically and
industrially particularly is very advantageous especially for human benefits.

Materials and Methods

(Week 1)

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Isolation Of Endospore Formers From The Soil

Collection of soil from a vegetative field was made. Soil sample of 1,0g was weighed
and added into 9ml of sterile Ringers saline solution (McCartney bottle). The solution
was then shook vigorously for 2 minutes, then aliquoted 1ml of suspension for dilution
series plating onto nutrient agar. The dilution of up to 10 -6 using the McCartney bottle
provided (9ml) was made. Using the spread plate technique 0,1ml aliquots of the
10-4, 10-5 and 10-6 dilutions was aseptically plated, this was done to duplicate the
nutrient agar plates which was then incubated at 30oC for 72 hours. McCartney bottles
was immersed in a water bath set to 80oC and maintained at this temperature for 15
minutes.

The heat treated soil suspension was used to determine aerobic endospore numbers
by making a use of 10-1, 10-2 and 10-3 dilution series using the McCartney bottles
provided (9ml).The spread plate technique was used to aseptically plate 0,1ml aliquots
of the 10-1, 10-2 and 10-3 dilutions to duplicate the nutrient agar plates, this was then
incubated at 30oC for 48-72 hours, the total number of endospore formers present in
1,0g of soil was determined and the obtained was compared to the total CFU count
for soil.

Endospore Staining

A loop-full of bacterial culture was aseptically transferred to a glass slide and a smear
preparation was made, the slide was allowed to air dry and fixed heated. The slide
was placed over a boiling beaker of H2O, flooded with 5% aqueous malachite green
and steamed for 10 minutes (it was ensured that the slide was kept flooded by
frequently adding fresh stain). There after, the slide was washed with running tap H2O
for 30 seconds, it was then counterstained with for 30 seconds with safranin for 60
seconds, washed in running H2O and blot dried. The prepared slide was then
examined under oil immersion (both endospores and free spores stain green;
vegetative cells stain red). The labelled, annotated diagrams of the morphological
arrangement of the endospores observed of each of the strains provided was made.

(Week 2)

Characterization of Unidentified Endospore Forming Isolates

Gram stain of each unknown isolates was carried out, (the reference strains would
have been viewed and drawn the week before). The x1000 magnification was used to

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view and record the cell size and morphology under oil immersion. The relative sizes
of each strain was compared, the aim was to distinguish between the different isolates
based on these criteria. The presence of endospores was notified, taking note of the
spore arrangement (terminal, sub-terminal and centre) within the cell, colony
morphology and colour was recorded. The following diagnostic tests were performed
to inoculate each of the isolates.

Voges-Proskauer Test (as modified for strains of Bacillus; detects acetoin

production)

Test material: 40% KOH, creatine. Phosphate inhibit acetoin production in some
strains and species of Bacilli, therefore medium indicated earlier is used, the medium
is dispensed in 5ml amounts. The tubes were inoculated with a small amount of culture
(a culture adjusted to an optimal density of 0,1 should be sufficient). The tubes were
incubated at 30oC, after 2, 4 and 6 days they were examined for acetoin production.
1ml was removed and 18 drops (0,5ml) of 40% KOH was added and a small amount
(one or two small crystals) of creatine. After 30 to 60 minutes a red colour should
develop for a positive reaction (meaning acetoin was produced).

Indole Test (to test for hydrolysis of tryptophan)

Test material: Kovacs reagent. The tryptone tubes were inoculated with a small
amount of culture and incubated for about 24 hours. About 10 drops (0,5ml) of Kovacs
reagent were added to each tube and shook gently, a deep red colour developed in
the presence of indole, negative reactions remained colourless or light yellow. NOTE:
Bacteria that contain the enzyme tryptophanase can hydrolyse tryptophan to its
metabolic products; indole, pyruvic acid, and ammonia. Of these products, indole is
not used and accumulates in the medium.

Citrate Test (Ability To Use Citrate As a Sole Carbon Source)

The citrate slant was inoculated stabbing the non-slant part of the slant and streaked
the slant portion of the citrate slant. Inoculated slant was incubated at 30 oC for 24
hours, a positive reaction was formation of blue (alkaline) colour on the green slant,
ad no colour change indicated negative reaction.

Starch Hydrolysis Test (To Test For The Presence Of Amylase Activity)

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Test material: Iodine solution. A starch plate was inoculated by streaking the bacterial
culture on the surface of the plate, the plate was then incubated at 30 oC (this increased
the growth rate of the culture). When the good growth was evident, the plate was
flooded with iodine solution (Grams iodine if available appears to work well). The
iodine combines with the available starch and form a deep purple colour, a positive
reaction was a clear zone surrounding the colony.

Casein Hydrolysis Test [To Illustrate The Ability To Hydrolyse Casein (Milk
Protein) Because Of The Presence Of Protease(s)]

A skim milk plate was streaked as in the case of the starch plate. Uninoculated skim
milk plate, the milk forms haziness on the plate. For those cultures whose colonies
had proteases present, clear area formed around the growth, this then constitutes a
positive reaction.

Carbohydrates Fermentation Test (Test For Ability To Utilize Glucose,

Arabinose, Xylose and Mannitol As Sole Carbon Sources)

The ability of a particular strain to use a specific sugar depends on a large extent on
the presence of necessary enzymes to utilize that carbon source, it also depends on
the presence of the necessary mechanisms to allow the sugar to be taken up by the
cell. When autoclaved in the presence of phosphates, especially in the mild alkaline
solutions, many sugars (particularly reducing sugars) will darken. Therefore all sugars
should be autoclaved separately and added aseptically after autoclaving, tubes
containing different specific sugars were inoculated with a small amount of culture to
avoid alkaline carryover from the inoculum. The tubes were then incubated at 30oC for
10 days, a positive response is generally seen after 2 or 3 days, both growth and acid
production was then observed, bromothymol blue changes to yellow below Ph 7.0-7.3.

Anaerobic Glucose Test (Test For Anaerobic Growth)

The anaerobic glucose broth medium (8ml) should be dispensed into a 16,5ml test
tube, the sterile tubes were heated before inoculation to drive off dissolved oxygen
and cooled to room temperature. A small amount of adjusted culture was added, then
covered the medium in the tube with a layer of sterile mineral oil, this was incubated
at 30oC and checked the growth and acid production after 7 days. A yellow colour
indicated positive reaction.

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Glucose-Ammonium Salts Medium Tests (Test For Acid Formation)

A small amount of adjusted culture was added to each tube, as with for anaerobic
respiration, incubated for 7 days, the growth and acid formation was noted. A yellow
colour indicates a positive reaction.

Discussion

In the whole entire world microorganisms have now brought several humans, and
other organisms desired benefits. Their rapid growth, simple genome
characteristically make them to be more special in industries because ease in which
they are manipulated. This is due to their diverse characteristics and functions they
have. Particularly in this experiment the unknown isolate was identified to be
B.cereus after performing Gram stain, endospore staining, characterization of
endospore forming isolates. The identified species is found in the soil due to its ability
to form endospores which enables it to survive harsh conditions for a long time. It is
also found in the gut of some mammals including humans where it compete with other
microorganisms such Salmonella and Campylobacter, which causes some disorder,
therefore the presence of B.cereus eradicates the possibility of these pathogens to
cause diseases (ecologically important).

Many of the substrates can be utilized by this species, especially organic compound
this implies a wide range of enzymes it possesses e.g the hydrolysis of starch indicates
the presence of the enzyme amylase. As per results, the range of temperatures it
grows at, tells us that B.cereus is a mesophilic, chemoorganotrophic as well as
heterotrophic organism. It can also grow on wide range of salt concentrations by
knowledge we know that organisms that grow in presence of certain salt concentration
are generally known as halotolerant. As the member of Bacilli, B.cereus are facultative
anaerobes that can form protective endospores which are highly resistant. Since this

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species consumed casein it is expected it possesses the enzyme lactase, pectinase,
and tryptophan hydrolase etc. As the experiment was performed, in the case of
anaerobic glucose test the errors that could arose was the contamination of aerobic
microbes should the aseptic technique not performed well, compensation for this was
to heat the medium to drive off the dissolved oxygen.

Conclusion

The unknown isolate was identified to be Bacillus cereus which is Gram-positive,

endospore former, heterotrophic organism having the ability to utilize wide range of
substrates both organic and inorganic due to its enzymatic secretion. Mesophilic
organism that can live human surviving temperature, can also be psychrophilic
because of endospore, therefore it inhabit wide range of habitats.

References

Saikia et al.,2011

Bergeys manual of firmicutes Schleifer and Kandler 1972

Alseth et.,al 2006

Sandle et.,al 2014

Brock book of microbiology 14th edition (Matigan et.,al)