Biochemical Testing

Streptococcus agalactiae
Otani, Aki
Palabao, Clarissa
Perez, Kryzzialyn
Pusung, Blaise
Reyes, Von
Rodriguez, Mico
Salayog, Kim

ABSTRACT

This paper is intended to identify an unknown organism through biochemical testing such
as T.S.I., L.I.A., Urea, M.R.V.P., L.I.A. and Citrate. The unknown organism was then concluded
as Streptococcus agalactiae, a gram positive aerotolerant anaerobic bacterium. Conclusion was
based on the results and data gathered from the experiment.

INTRODUCTION
Streptococcus agalactiae is a member of the gastrointestinal normal flora in some
humans and can spread to secondary sites - including the vagina in 10–30% of women. This is of
clinical importance: S. agalactiae can be transferred to a neonate passing through the birth canal
and can cause serious group B streptococcal infection. In the western world, S. agalactiae is the
major cause of bacterial septicemia of the newborn, which can lead to death or long-term
sequelae. S. agalactiae invades via alveolar and pulmonary epithelial cells; newborns are
especially susceptible to infection because they lack alveolar macrophages to prevent invasion.
Newborn GBS disease is separated into early-onset disease occurring on living days 0–7 and
late-onset disease which starts on days 7–90. Early-onset septicemia is more prone to be
accompanied by pneumonia, while late-onset septicimia is more often accompanied by
meningitis. S. agalactiae neonatal meningitis doesn't present with the hallmark sign of adult
meningitis, a stiff neck; rather, it presents with nonspecific symptoms, such as fever, vomiting
and irritability, and can consequently lead to late diagnosis. Hearing loss can be a long-term
sequela of GBS-meningitis. Infection with GBS is the cause of some instances of stillbirth.
S. agalactiae is present in up to one-third of women of childbearing age, and 1.8 cases
per 1000 live births will be affected by group B streptococcal infection. In the elderly or persons
with compromised immune systems, septicemia or other serious infections are seen. This can
also occur during pregnancy or maternity.
Biochemical testing necessitates the determination of different parameters, and the
identification of the main biological chemical compounds, by using molecular and biochemical
tools.
 1. Koser’s citrate or Simmon’s citrate to test
for the ability of bacteria to ferment citrate.
When citric acid or sodium citrate is in
solution, it loses a proton or sodium ion
to form a citrate ion. Bacteria with citrate
lyase can break down citrate to form
pyruvate. Pyruvate can be further reduced
in fermentation.
Purpose of performing the citrate test
o Tests for the ability of bacteria to convert
citrate (an intermediate of the Kreb’s cycle)
into oxaloacetate (another intermediate of the
Kreb’s cycle)
o of Simmon citrate include
o sodium citrate as the carbon source
o monoammonium phosphate as the nitrogen source
o and bromthymol blue indicator that changes to blue when medium turnsalkaline, which
means  (+) result.

When bacteria uses citrate(carbon) and ammonium(nitrogen), medium turns alkaline as ammonia
is produced from ammonium.

2. MRVP test is able to differentiate

between organisms that produce large
amounts of acid and organisms that only
produce neutral content(acetoin)
M-Methyl Red. VP- Voges -Proskauer. Methyl
Red is different from Phenol Red that we
mention earlier. It has a pH of 4.4-6. Hence, it
changes colour at this range.
For MR test,  if  organic acid is produced here,
the (+) result is just a no change in colour of
Methyl Red. RED remains.
if neutral acetoin is produced here, the (-) result
is a change in colour
fromRED to YELLOW because at pH 7, the
indicator will change its colour.
To test for the presence of acetoin, we use VP
test where potassium hydroxide and α naphthol
are added.
The upper of the medium will turn red. (+) If the medium turns light brown, it is a (-) result.
Please note that production of acetoin is also affected by the duration of incubation, hence, false
negative results may be observed.
 3. Urease Test breaks down urea into ammonia (NH3)
& carbon dioxide (CO2).
 
Properties of urea medium
Since there’s phenol red pH indicator, pH indicator changes
from yellow to bright pink if NH3 is produced.
At more than pH 6.8, the colour change represents a (+) result
Proteus vulagris is one bacteria that produces urease to break
down urea.
This test is used to identify bacteria capable of
hydrolyzing urea using the enzyme urease. It is
commonly used to distinguish the genus Proteus from
other enteric bacteria. The hydrolysis of urea forms the
weak base, ammonia, as one of its products. This weak
base raises the pH of the media above 8.4 and the pH
indicator, phenol red, turns from yellow to pink. Proteus
mirabilis is a rapid hydrolyzer of urea (center tube pictured here). The tube on the far
right was inoculated with a urease negative organism and the tube on the far left was
uninoculated.
4. Sulfur Indole Motility Media (SIM) This is a differential medium. It tests the ability of
an organism to do several things: reduce sulfur, produce indole and swim through the
agar (be motile). SIM is commonly used to differentiate members of Enterobacteriaceae.

Sulfur can be reduced to H2S (hydrogen sulfide) either by catabolism of the amino acid
cysteine by the enzyme cysteine desulfurase or by reduction of thiosulfate in anaerobic
respiration. If hydrogen sulfide is produced, a black color forms in the medium. Proteus
mirabilis is positive for H2S production. The organism
pictured on the far left is positive for hydrogen sulfide
production.

Bacteria that have the enzyme tryptophanase, can

convert the amino acid, tryptophane to indole. Indole
reacts with added Kovac’s reagent to form rosindole
dye which is red in color (indole +). Escherichia coli is
indole positive. The organism pictured second from
left is E. coli and is indole positive.

SIM tubes are inoculated with a single stab to the bottom of the tube. If an organism is motile
than the growth will radiate from the stab mark and make the entire tube appear
turbid. Pseudomonas aeruginosa and the strain
of Proteus mirabilis that we work with are motile.

5. SIM (Sulfur Indole Motility Test) is a

differential medium used to determine
whether an organism is equipped with
flagella and thus capable of swimming away
 from a stab mark. The results of motility agar are often difficult to interpret. Generally, if
the entire tube is turbid, this indicates that the bacteria have moved away from the stab
mark (are motile). The organisms in the two tubes pictured on the right are motile. If,
however, the stab mark is clearly visible and the rest of the tube is not turbid, the
organism is likely nonmotile (tube pictured on the left).

6. Lysine iron agar (LIA) slants test organisms for the ability to deaminate lysine or
decarboxylate lysine.  Lysine deamination is an aerobic process which occurs on
the slant of the media.  Lysine decarboxylation is an anaerobic process which occurs in
the butt of the media.

LIA slants contain lysine, glucose, peptones, bromcresol purple (pH indicator), sodium
thiosulfate and ferric ammonium citrate. If the organism has the ability to decarboxylate lysine, it
produces an amine end-product which reacts with the pH indicator to give a purple color in
the butt of the tube. (Negative decarboxylation:  yellow butt). 

If the organism has the ability to deaminate lysine, the ammonia produced will react with the
ferric ammonium citrate to produce a dark red color on the slant of
the tube. (Negative deamination:  purple slant). Organisms which
produce hydrogen sulfide gas will exhibit a black precipitate in the
butt of the tube.

 Tube 1:  Positive decarboxylation (butt), negative deamination

(slant)

Tube 2:  Negative decarboxylation (butt), positive deamination

(slant)

[ CITATION famsbc \l 13321 ]

MATERIALS AND METHODS

(FROM LAB MANUAL)

RESULTS AND DISCUSSION

Figure 1. Biochemical Test. Prepared agar used in identifying the unknown organism.
 Figure 1 shows the different prepared agar in five different test tubes such as Citrate, TSI,
SIM, LIA, Urea agars and MRVP broth. An unknown organism was streaked in these five test
tubes using different streaking techniques. The preparation and incubation of the prepared agars
were performed beforehand in order to save time and effort. Incubation period of cultured test
tubes is 24 hours at 37.5C temperature.
1. Citrate Test

INSERT OBSERVATIONS HERE and from LAB

MANUAL ( DO THE SAME FOR THE NEXT FIVE
TESTS) (YUNG MISMONG NAKITA NILA KIM)

Figure 2 Citrate Test. No visible colony was

present.

2. TSI (Triple Sugar Iron)

Figure 3 TSI. Shows forming of colonies.

 3. SIM

Figure 4. No Motility was present but a change of color was observed.

4. LIA

Figure 5. LIA. No colony formation.

 5. Urea

Figure 6. Urea test.

6. MRVP Test

CONCLUSION

Figure 7 tHIS ONE TOO DI KO ALAM

Figure 8. DI KO ALAM THIS ONE
 Biochemical Test
Citrate TSI LIA SIM Urea MRVP

PAKI LAGYAN PO KUNG POSITIVE OR NEGATIVE

Based on the data and results obtained from the experiment, we therefore conclude that the
unknown organism is Streptococcus agalactiae. PAKI DUGTUNGAN PO. BASE po TO SA
OBSERVATIONS NA NAKITA NILA KIM.

References:

Famsbc. (2009, July 30). Biochemical tests- MICROBIOLOGY. Retrieved October 1, 2012, from Wordpress:
http://famsbc.wordpress.com/

LAGAY NYU DITO REFERNCE LAB MANUAL SA MICRO DI KO DALA BOOK KO SORRY,
I SOULD’VE FILLED UP THE RESULTS AND DISCUSSION PATI CONCLUSION IF I HAVE
MY BOOK WITH ME. :(