EXPERIMENT : Determination of specified media for cultivation bacteria

Control of Microbial Growth

Identification of bacteria using RapID ONE System
OBJECTIVES
1. To use of specified media to cultivate bacteria
2. To identify the characteristic of bacteria
3. To study the structure of bacteria formed in different types of media
4. To identify growth of bacteria when subjected to hand sanitizer
5. To study the antibiotic resistance of the bacteria
6. To study the effect of heat on bacteria
7. To identify bacteria using Rapid One system
INTRODUCTION
Selective media is a type of media that selects for the growth of one type of
organism, while inhibiting the growth of others. An example of Selective media is
MacKonkey Agar. MacConkeys Agar is selective for gram-negative bacteria, while inhibiting
the growth of gram-positive bacteria.
Differential media contain compounds that allow groups of microorganisms to be
visually distinguished by the appearance of the colony or the surrounding media, usually on
the basis of some biochemical difference between the two groups. Blood agar is one type of
differential medium, allowing bacteria to be distinguished by the type of hemolysis
produced. Some differential media are also selective, i.e., most of the standard enteric agars
such as MacConkey and EMB agars, which are selective for gram-negative coliforms and
which differentiate lactose-fermenting and non-lactose-fermenting bacteria.
An enriched media enhances growth-promotion in certain organisms. These
organisms require ingredients like blood and glucose in order to grow. Enriched media
contains these ingredients and assist in helping organisms grow.
The control of microbial growth may involve sterilization, disinfection, antisepsis,
sanitization, or degerming. Sterilization is the destruction of all forms of microbial life, with

particular attention to bacterial spores. Disinfection and antisepsis both refer to destruction
of microbial pathogens, although some organisms, such as bacterial spores, may remain
alive. Disinfection refers to the destruction of pathogenic organisms on an inanimate
(lifeless) object, such as a table-top, while antisepsis refers to that destruction on a living
object, such as the skin surface. Sanitization refers to the reduction in the number of
pathogens to a level deemed safe by public health guidelines.
The RapID ONE System is a qualitative micromethod employing conventional and
chromogenic substrates for the identification of medically
important Enterobacteriaceae and other selected oxidase-negative, Gram-negative bacilli
isolated from human clinical specimens.

One-step inoculationdecreased prep time for increased productivity

Manual microbial identification made easyno oil, no pipetting, no 24-hr incubation

Four-hour incubation*quicker time-to-result for a faster response

Common procedurelessened materials usage saves time and money

Visible color reactionsreduced subjectivity decreases repeat testing

Better reportingmore coverage, advanced updates

*Two-hour incubation for RapID SS/u.
A clear plastic tray contains reagent impregnated wells, allowing simultaneous

inoculation of each cavity with a predetermined volume of inoculums. A suspension of test

organism in RapID Inoculation Fluid is used as the inoculums which rehydrates and initiates
test reactions.
After incubation, each cavity is examined for reactivity by noting the development of a
color. In some cases, reagents must be added to the cavities to provide a color change. The
resulting pattern of positive and negative scores is used as the basis for identification of the
test isolate by comparison of test results to reactivity patterns stored in a database or
through the use of a computer-generated Code Compedium (ERIC).

PROCEDURE
Experiment 1 : Determination of specified media for cultivation bacteria
1. A NA plate was obtained, on outside of the bottom half (the part that contains the
media) label it
2. Inoculating was sterilized loop and let it cooled
3. Open source culture and the mouth of the tube was flamed
4. The sterile loop was deepen into liquid culture containing bacteria
5. Loop from source culture was removed, flamed mouth of tube and replaced cap
6. The loopful of the organism in the area of the nutrient agar plate and streak the
culture back and forth
7. Loop flamed and cooled
8. Turn plate of a turn, the loop passed once over the previously streaked area and
proceeded to streak a new area of the plate without entering the first 1\3 section
9. The last two steps was repeated until the plate is completely streaked
10. Lid was replaced, sterilized the loop, incubated plate upside down (agar side up)
11. Agar plate was divided to two parts labeled A and B.
12. Steps 1 to 10 to each division of A and B without mixing the two parts.
13. The steps were repeated using different agar.

Experiment 2 : Control of Microbial Growth

A. USE OF HAND SANITIZERS
1. Divided the plate into 4 column and label each column with Hand before wash,
Hand after wash, Phone before apply sanitizer and Phone after apply
sanitizer.
2. Using a cotton swap, gently rub it on the palm, nails and between fingers.
3. Streaks the cotton swap on the column labeled Hand before wash.
4. Next, wash hand using sanitizer provided. You must follow the correct procedure
of washing hand.

5. Once again, using new cotton swap, gently rub it on the palm, nails and between
fingers on the hand that have been washed.
6. Streaks the cotton swap on the column labeled Hand after wash.

B. USE OF SANITIZER ON CELLULAR PHONE

1. Using a cotton swap, gently rub it on the phone screen surface.
2. Streaks the cotton swap on the column labeled Phone before apply sanitizer.
3. Next, appy sanitizer on phone screen using tissue paper.
4. Once again, using new cotton swap, gently rub it on phone screen surface.
5. Streaks the cotton swap on the column labelled Phone after apply sanitizer.

C. PREPARATION OF INOCULUM
1. Open the bacteria plate that have been given to you and carefully smear the
bacteria colony using cotton swap.
2. Open the Mueller-Hinton broth tube and insert the cotton swap into the
solution.
3. Stir the Mueller-Hinton broth using the cotton swap until a cloudy solution is
produced.
D. DETERMINATION OF ANTIBIOTIC RESISTANCE
1. Divide two plate of agar into 5 parts.
2. Using cotton swap, immerse it into the inoculums prepared.
3. Remove the cotton swap and streak on the agar surface on every parts of the
agar.
4. Place the antibiotic given using forceps one part each

E. UV TREATMENT
1. Divide the plate into half and label 25+5 minutes and 5 minutes.
2. Using cotton swap, immerse it into the inoculums prepared.
3. Remove the cotton swap and streak on the agar surface on the 25+5 minutes
column.
4. Place it in UV chamber for 25 minutes.
5. After 25 minutes, take the plate.

6. Using cotton swap, immerse it into the inoculums prepared.

7. Remove the cotton swap and streak on the agar surface on the 5 minutes
column.
Experiment 3: Interpretation of bacteria using RapID ONE System
1. Peel of the label lid over the reaction cavities by pulling the lower right hand tab up
and to the left.
2. Add 2 drops of RapID ONE Reagent to cavities 15 (PRO) through 17 (PYR).
3. Read and score cavities 1 (URE) through 18 (ADON) from left to right using the
interpretation guide presented in Table 1. Record test scores in the appropriate
boxes on the report form using the test code above the box for the bifunctional test.
4. Add 2 drops of RapID Spot Indole Reagent to cavity 18 (ADON/IND).
5. Allow at least 10 seconds but no more than 2 minutes for color development.
6. Read and score test cavity 18 (IND). Record the score in the appropriate box of the
report form.
7. Refer the microcode obtained on the report form in the ERIC (Electronic RapID
Compendium) for the identification.
OBSERVATION AND RESULTS
Experiment 1 : Determination of specified media for cultivation bacteria
MEDIA

AMOUNT OF
GROWTH

APPEARANCE OF APPEARANCE OF REMARKS

COLONY
MEDIA

EMB

Nutrient Agar

+++

White

No change

Large
Rough
Dull
Non
pigmented

BHI

+++

Cream

No change

No change

Large
Rough
Dull
Non
pigmented
NA + 5% human
blood

+++

White
Large
Rough
Dull
Non
pigmented

Mc Conkey

MSA

NA + VANCO

TCBS

ORSA

MRS

BPA

Experiment 2: Control of microbial growth

A. USE OF HAND SANITIZERS & USE OF SANITIZER ON CELLULAR PHONE
COLUMN
Hand before wash
Hand after wash
Phone before apply sanitizer
Phone after apply sanitizer

OBSERVATION
Colony of bacteria grow
No growth
Colony of bacteria grow
No growth

B. DETERMINATION OF ANTIBIOTIC RESISTANCE

ANTIBIOTIC
CRO, ceftriaxone, 30s
CN, GENTAMICIN, 10s
TZP, piperacillin-tazobactam 10:1, 110 s
AMC amoxicillin-clavulanic acid 2:1, 30
SAM, ampicillin-sulbactam, 20g
LEV, Levofloxacin, 5 g
FOF, Fosfomycin, 50g
NET, netilmicin, 30 g
TE, Tetracyclines
N, Neomycin

DIAMETER
1.8
2.8
3.8
1.6
1.7
3.4
3.4
2.8
2.6
2.4

C. UV TREATMENT
COLUMN
25+5 minutes
5 minutes

OBSERVATION
Growth of bacteria
No growth

Experiment 3 : Interpretation of bacteria using Rapid One system

DISCUSSION
Experiment 1 : Determination of specified media for cultivation bacteria
In nutrient agar, bacteria grow tremendously fast when supplied with an abundance of
nutrients. Different types of bacteria will produce different-looking colonies, some colonies
may be colored, some colonies are circular in shape, and others are irregular. The
characteristics of a colony (shape, size, pigmentation, etc.) are termed the colony
morphology. Colony morphology is a way scientists can identify bacteria. Each distinct
circular colony should represent an individual bacterial cell or group that has divided
repeatedly. Being kept in one place, the resulting cells have accumulated to form a visible
patch. Most bacterial colonies appear white, cream, or yellow in color, and fairly circular in
shape. Yeast colonies generally look similar to bacterial colonies. Some species, such
as Candida, can grow as white patches with a glossy surface.
Brain Heart Infusion Agar (BHI Agar) is an enriched non-selective medium for the isolation
and cultivation of most anaerobic bacteria and other fastidious microorganisms. The basic
nutritive properties are brain heart infusion from solids as well as meat peptones,with the
addition of yeast extract. This medium is supplemented with hemin and vitamin K1 as
growth factors for most anaerobic bacteria. This medium is prepared, dispensed, stored
and packaged under oxygen-free conditions to prevent the formation of oxidized products
prior to use. This medium should support good growth of many fastidious and nonfastidious anaerobes isolated from clinical specimens If the nutritive/inhibitory capacity of
this medium is to be tested for performance, it is recommended that the

following ATCCorganisms be evaluated for growth/inhibition. Physical appearance: BHI agar

should appear opaque to translucent and yellow in color.
Organism

Expected
Growth

B. fragilis

24 hrs

C. perfringens

24 hrs

P. anaerobius

24 hrs

S. aureus

24 hrs

E. coli

24 hrs

If a bacterial growth medium is selective, that means that it grows only certain types of
microbes while inhibiting the growth of other types of microbes. Blood agar is an enriched
medium that provides an extra rich nutrient environment for microbes. Therefore, BAP is
not a selective growth medium, since it supports the growth of a wide range of organisms. A
growth medium is considered differential if, when specific microbes are present, the
medium or bacterial colonies themselves exhibit a color change that provides information
about their identity.
Blood agar (BAP) is a differential growth medium which microbiologists use to distinguish
clinically significant bacteria from throat and sputum cultures. Certain bacteria produce
exotoxins called hemolysins, which act on the red blood cells to lyse, or break them down.
Blood agar is usually inoculated from a patients throat swab, because the medical
laboratory is trying to detect the presence of Group A beta hemolytic Streptococci (a Grampositive round shaped bacteria that causes beta-hemolysis on blood agar.) The major
human pathogen in this group is Streptococcus pyogenes, the causative agent of strep
throat. Normal throat flora will exhibit alpha or gamma hemolysis.
Blood agar contains general nutrients and blood. It is useful for cultivating fastidious
organisms and for determining the hemolytic capabilities of an organism.
Some bacteria produce exoenzymes that lyse red blood cells and degrade hemoglobin;
these are called hemolysins. Bacteria can produce different types of hemolysins. Betahemolysin breaks down the red blood cells and hemoglobin completely. This leaves a clear
zone around the bacterial growth. Such results are referred to as -hemolysis (beta
hemolysis). Alpha-hemolysin partially breaks down the red blood cells and leaves a greenish
color behind. This is referred to as -hemolysis (alpha hemolysis). The greenish color is
caused by the presence of biliverdin, which is a by-product of the breakdown of

hemoglobin. If the organism does not produce hemolysins and does not break down the
blood cells, no clearing will occur. This is called -hemolysis (gamma hemolysis).
The hemolysins produced by streptococci perform better in an anaerobic
environment. Because of this, it is standard procedure to streak a blood plate and then stab
the loop into the agar to provide an area of lower oxygen concentration where the
streptolysins can more effectively break down the blood cells.
BHI agar a solid medium which contains the highly nutritious infusions recommended for
the cultivation of most anaerobic bacteria and other fastidious microorganisms. Brain Heart
Infusion Agar may be recommended for the cultivation of streptococci, Neisseria and other
fastidious organisms. There are present of growth of colonies on this media so it is assume
that BHI is an enriched non-selective medium for the isolation and cultivation of most
bacteria.
MacConkey Agar (MAC) is a selective and differential medium designed to isolate and
differentiate enterics based on their ability to ferment lactose. Bile salts and crystal violet
inhibit the growth of Gram positive organisms. Lactose provides a source of fermentable
carbohydrate, allowing for differentiation. Neutral red is a pH indicator that turns red at a
pH below 6.8 and is colorless at any pH greater than 6.8.
Organisms that ferment lactose and thereby produce an acidic environment will appear pink
because of the neutral red turning red. Bile salts may also precipitate out of the media
surrounding the growth of fermenters because of the change in pH. Non-fermenters will
produce normally-colored or colorless colonies.
Mannitol salt agar (MSA) is both a selective and differential medium used in the isolation of
staphylococci. It contains 7.5% sodium chloride and thus selects for those bacteria which
can tolerate high salt concentrations. MSA also distinguishes bacteria based on the ability to
ferment the sugar mannitol, the only carbohydrate in the medium. Staphylococci can
withstand the osmotic pressure created by 7.5% NaCl, while this concentration will inhibit
the growth of most other gram-positive and gram-negative bacteria (14). Additionally, MSA
contains mannitol and uses phenol red as a pH indicator (pK = 7.8) in the medium. At pH
levels below 6.9, the medium is a yellow color. In the neutral pH ranges (6.9 to 8.4) the color
is red; while above pH 8.4, the color of phenol red is pink (11). When mannitol is fermented
by a bacterium, acid is produced, which lowers the pH and results in the formation of a
yellow area surrounding an isolated colony on MSA. A nonfermenting bacterium that
withstands the high salt concentration would display a red to pink area due to peptone
breakdown (15).
BD Baird-Parker Agar contains the carbon and nitrogen sources necessary for growth.
Glycine, lithium chloride and potassium tellurite act as selective agents. Egg yolk is the
substrate to detect lecithinase production, and, in addition, lipase activity. Staphylococci

produce dark gray to black colonies due to tellurite reduction; staphylococci that produce
lecithinase break down the egg yolk and cause clear zones around respective colonies. An
opaque zone of precipitation may form due to lipase activity. The medium must not be used
for the isolation of staphylococci other than S. aureus.
Experiment 2: Control of microbial growth
The control of microbial is necessary in order to minimize infection and disease. In this
experiment we will see the action of sanitizer in market in the reduction of bacteria and its
effectiveness. From the experiment, we proved that the use of sanitizer is effective in order
to reduce the bacteria population. The brand and price of hand sanitizer is unimportant as
lower price and not popular brand can also use to reduce bacteria population. Sometimes,
branded sanitizers are not that effective as the non-branded. It is important to check the
material use in the sanitizers.
Difference bacteria have difference resistance towards difference antibiotic. In this
experiment we will determine the antibiotic resistance of the bacteria by measuring the
diameter of region of hydrolyzation of bacteria by certain antibiotic. There are 10 different
antibiotic use I this experiment.
Ceftriaxone injection is used to treat certain infections caused by bacteria such as
gonorrhea (a sexually transmitted disease), pelvic inflammatory disease (infection of the
female reproductive organs that may cause infertility), meningitis (infection of the
membranes that surround the brain and spinal cord), and infections of the lungs, ears, skin,
urinary tract, blood, bones, joints, and abdomen. Ceftriaxone injection is also sometimes
given before certain types of surgery to prevent infections that may develop after the
operation. It works by killing bacteria. Bacteria 12 have low resistance toward ceftriaxone.
Gentamicin is an aminoglycoside antibiotic composed of a mixture of related gentamicin
components and fractions and is used to treat many types of bacterial infections,
particularly those caused by Gram-negative organisms. In this experiment, bacteria 12 have
medium resistance toward gentamicin.
Piperacillin/tazobactam is a combination antibiotic containing the extendedspectrum penicillin antibiotic piperacillin and the -lactamase inhibitor tazobactam The

combination has activity against many Gram-positive and Gram-negative pathogens

andanaerobes, including Pseudomonas aeruginosa. Bacteria 12 show high resistance
towards TZP.
The combination of amoxicillin and clavulanic acid is used to treat certain infections caused
by bacteria, including infections of the ears, lungs, sinus, skin, and urinary tract. Amoxicillin
is in a class of medications called penicillin-like antibiotics. It works by stopping the growth
of bacteria. It works by preventing bacteria from destroying amoxicillin. These bacteria have
lowest resistance towards amoxilin-cluvanic acid.
Ampicillin/sulbactam is a combination of the common penicillinderived antibiotic ampicillin and sulbactam, an inhibitor of bacterialbeta-lactamase. Two
different forms of the drug exist. Ampicillin/sulbactam is used to treat infections caused by
bacteria resistant to beta-lactam antibiotics. Sulbactam blocks the enzyme which breaks
down ampicillin and thereby allows ampicillin to attack and kill the bacteria. The bacteria 12
have low resistance to SAM.
Levofloxacin is used to treat certain infections such as pneumonia, chronic bronchitis and
sinus, urinary tract, kidney, prostate (a male reproductive gland), and skin infections.
Levofloxacin is also used to prevent anthrax (a serious infection that may be spread on
purpose as part of a bioterror attack) in people who may have been exposed to anthrax
germs in the air. It works by killing bacteria that cause infections. Bacteria 12 have high
resistance toward LEV.
Fosfomycin is an antibiotic used to treat infections of the urinary tract. Bacteria 12 have
same resistance towards fosfomycin as its resistance towards levoflaxin.
Netilmicin is a member of the aminoglycoside family of antibiotics. These antibiotics have
the ability to kill a wide variety of bacteria. Netilmicin is not absorbed from the gut and is
therefore only given by injection or infusion. It is only used in the treatment of
seriousinfections particularly those resistant to gentamicin. Bacteria 12 shows medium
resistance towards gentamicin.
Tetracycline, is used to treat bacterial infections, including pneumonia and other respiratory
tract infections; acne; infections of skin, genital and urinary systems; and the infection that

causes stomach ulcers (Helicobacter pylori). It also may be used as an alternative to other
medications for the treatment of Lyme disease and for the treatment and prevention of
anthrax (after inhalational exposure It works by preventing the growth and spread of
bacteria. This antibiotic have medium resistance on bacteria 12.
Neomycin, an antibiotic, is used to prevent or treat skin infections caused by bacteria. It is
not effective against fungal or viral infections. Bacteria 12 shows medium resistance
towards this antibiotic.
In UV treatment analysis, the bacteria should grow on both columns. But, the bacteria only
grow on the column that is treated with 25 minutes UV. This means that the bacteria 12 are
heat resistance. It can still grow even if subjected to high temperature surroundings.
Experiment 3 : Interpretation of bacteria using RapID ONE System
In this experiment, we obtained the different colour for different test. URE test is negative
as there is no development of red or violet colour. As for ADH, ODC and LDC, there is a
development of distinct, bright purple colour and scored as positive. TET and LIP shows
negative feedback as the solution form is red and orange. KSF and SBL show positive result
with the formation of orange solution. GUR, ONPG, GLU, XYL and NAG also shows the
negative result because of the formation of very faint yellow is observed. MAL form orange
solution and scored as negative. PRO, GGT and PYR after treated with Rapid One reagent,
they form clear, yellow, orange and very pale pink and the result is negative. As for ADON,
dark red-orange is form giving it a negative result. IND after treated with Rapid spot indole
reagent form a orange solution giving it a negative result.
Based on the result, the referral number obtained is 6160000 and we get the named of
bacteria as Salmonella choleraesuis but the bacteria given to us is Bacillus cereus. There is
some limitation that may cause different result obtained.
1. The use of the RapID ONE System and the interpretation of results require a
competent laboratorian who is trained in general microbiological methods and who
should judiciously make use of knowledge, experience, specimen information, and
other pertinent procedures before reporting the isolate identity obtained using the
RapID ONE System.

2. Specimen source, oxidase reaction, Gram stain characteristics, and growth on

selective agars should be considered when using the RapID ONE System.
3. The RapID ONE System must be used with pure cultures of test organisms. The use
of mixed microbial populations or direct testing of clinical material without culture
will result in aberrant results.
4. The RapID ONE System is designed for use with the taxa listed in the RapID ONE
Differential Chart. The use of organisms not specifically listed may lead to
misidentifications.
5. Expected values listed for RapID ONE System tests may differ from conventional
test results or previously reported information.
6. The accuracy of the RapID ONE System is based upon the statistical use of a
multiplicity of specially designed tests and an exclusive, proprietary database. The
use of any single test found in the RapID ONE System to establish the identification
of a test isolate is subject to the error inherent in that test alone.
CONCLUSION
In this experiment, using nutrient agar, it shows the bacterial growth with white colonies.
These colonies have appeared in a rough, dull, large and non-fermented structure. Most
bacteria colonies can easily grow in the nutrient agar. Next, in Brain-Hearted Infusion (BHI)
agar, our bacteria have formed cream colonies with circular shape. This shows our bacteria
have the ability to grow under anaerobic condition. Blood agar usually used to distinguish
chemically significant bacteria from throat sputum cultures. These bacteria can grow in
blood agar media but it cannot hydrolyze the blood agar.
These bacteria shows a high resistance towards TZP or Piperacillin/tazobactam while shows
the lowest resistance towards AMC or amoxcillin-cluvanic acid.
The bacteria are heat resistance and can grow well under hot condition.
The referral number is 6160000 and we get the named of bacteria as Salmonella
choleraesuis but the bacteria given to us is Bacillus cereus.

REFERENCES
1) https://www.google.com.my/?gws_rd=cr&ei=yKRoU-yzOcHJuASKpoDwDA#q=blood+agar
2) http://himedialabs.com/TD/M001.pdf
3) http://www.anaerobesystems.com/Home/pras-mono-plated-media/Brain-HeartInfusion-Agar
4) http://www.austincc.edu/microbugz/mannitol_salt_agar.php
5) http://amrita.vlab.co.in/?sub=3&brch=73&sim=213&cnt=1
6) www.thermoscientific.com
7) aac.asm.org
8) www.lucron.eu
9) www.webmd.com
10) www.ann-clinmicrob.com
11) www.cliffsnotes.com