FACULTY OF MEDICINE

PRACTICAL
MICROBIOLOGY-LOG BOOK

MBBS, Batch 27

CANDIDATES I.D. NUMBER

Sahnusha Sryen Samudram M21100771

Unit of Microbiology
Faculty of Medicine
AIMST UNIVERSITY
MCVS 65104

Sahnusha Sryen Samudram M21100771

Refer to- AIMST Microbiology Student’s Lab Handbook, Harcharan Singh,

Rajesh PK et al, 2005

(Revised-January 2021, Updated July 2022)

For any clarifications, please contact Prof. P K Rajesh, email: rajesh@aimst.edu.my

 SYNOPSIS OF YEAR 1 MICROBIOLOGY MCVS PRACTICAL

Case-1
A 70-year- old male was admitted to the hospital after suffering extensive third-degree burns. Three
days later, he spiked a fever, and there was pus oozing from the skin wound that had a blue-green
color. On examination, he had a temperature of 39⁰C, tachypnea(R>24/min) and tachycardia
(P>90/min) that suggested systemic inflammatory response syndrome (SIRS). There were obvious
signs of microbial infection, so a diagnosis of sepsis was made.

The pus and blood samples were sent for laboratory investigation (Microscopy, culture and ABST)

Laboratory Findings

Sample Blood, pus

Gram-stained Showed gram negative bacilli and pus cells.
smear of the pus

Pus and Blood Blue-green pigment on ordinary nutrient agar

Culture Non-lactose-fermenting colonies on MacConkey’s agar
Gram staining of Gram-negative rods
isolated organism
Biochemical test oxidase-positive

Antibiotic Sensitive (S) : piperacillin, piperacillin+ tazobactam,

Sensitivity Ticarcillin+ tazobactam, Imipenem
test(ABST) Resistant (R) : gentamicin, amikacin, ciprofloxacin

Interpretation: Pseudomonas aeruginosa is present. Blue-green pigment formed on nutrient agar and
biochemical test shows oxidase positive.
Case-2
A 34-year-old man presented with the complaints of sudden onset of fever, shaking chill and
shortness of breath at rest. On examination, his temperature was 39°C, blood pressure 110/60 mm of
Hg, pulse 104/min, respiratory rate 18/min. Janeway lesions were found on the palms. A new
murmur consistent with tricuspid insufficiency was heard. Needle tracks were seen on both
forearms. Echocardiography showed vegetations on tricuspid valve.

Blood samples were taken and sent to laboratory for further evaluation.

Laboratory Findings
Sample Blood

Culture Golden-yellow colonies on Nutrient agar and Mannitol salt agar.

β-hemolytic colonies on blood agar

Gram staining of Gram-positive cocci in grapelike clusters

isolated organism

Biochemical test Positive for both catalase and coagulase test

Antibiotic Sensitive (S) : Vancomycin, Ceftriaxone, Oxacillin, Levofloxacin

Sensitivity Resistant (R) : Penicillin G, Tetracycline
test(ABST)

Interpretation: Staphylococcus aureus because of golden-yellow colonies on nutrient agar and B-

hemolytic colonies on blood agar, also, positive coagulase test confirmed my interpretation

Case-3
A 40-year-old man with persistent fever and night sweats for the past 4 weeks suddenly developed
increasing fatigue and shortness of breath. The patient had history of rheumatic fever when he was
15 years old. Two molar teeth were extracted about 12 weeks back without any chemoprophylaxis.
On examination, he was febrile. His spleen was palpable. Splinter haemorrhage and Osler’s node
were found in the right thumb and right toe respectively. A loud holosystolic murmur was heard
over the precordium. He was anaemic, and his WBC count was 13.5×10⁹/L. Echocardiogram
revealed vegetation in mitral valve. Blood samples were taken and sent to laboratory for further
evaluation.

Sample Blood

Culture Green (alpha) hemolysis on blood agar

Gram staining of Gram-positive cocci in chains

isolated organism

Biochemical test Negative for catalase test

Not inhibited by optochin

Not bile-soluble
Antibiotic Sensitive (S) : Amoxycillin , gentamicin and vancomycin,
Sensitivity test
(ABST) Resistant (R) : Penicillin G, Tetracycline

Interpretation: Streptococcus viridans, mainly because of the alpha hemolysis on blood agar is not
inhibited by
Optochin.
Diagnosis of Bacterial Infection
microscopy

cu l t u re

sen si t i vi t i es b y d isc diffusion

methods,
br ea kp oi nt s or
MICs Serodiagnosis D N A technologies

Protocol in Bacteriology
DAY 1
Microscopy
G r a m stain, W e t m o u n t , D a r k g r o u n d ,
N e g a t i v e staining, sp eci al sta ins
C ul t ur e
P u r e culture isolation- s el ect a g a r a c c o r d i n g
to g r a m stai n findings. B l o o d a ga r , Nutri ent
ag ar , M a c C o n k e y
R a p i d tests – a n t i g e n de tecti on m e t h o d s

MBBS Batch 27
 P r o t o c o l in b a c t e r i o l o g y
DAY 2
R e a d i n g t he cul t ure to i d e n tify b a cteri a
B i o c h e m i c a l tests t o c o nf i r m ident i ty
P utti ng u p t he a n t i b i o g r a m

DAY 3
R e a d i n g t he a n t i b i o g r a m

MBBS Batch 27
MCVS 65104
Describe your personal experience (including what you
did/what was learnt/new information/how it would have
been better etc) during the MCVS microbiology practical:

During our first offline practical, I would say I had a lot of fun because I understood
everything Dr Shandra was saying to us. We were able to ask her questions that we had
been avoiding during our online sessions; it was truly interactive. I finally had the
opportunity to watch different organisms and put them through some tests, such as the
catalyst test. In addition, I was able to watch and do the oxidase and the culture medium.
It was thrilling to observe the evolution of each test. Dr. Shandra was very patient with us
as she guided us through the entire session. Overall, it was a positive experience, and I
am looking forward to our next system's practical sessions
MBBS Batch 27
Explain the term infective endocarditis, native valve endocarditis (NVE),
prosthetic valve endocarditis (PVE) and intravenous drug abuse (IVDA)
endocarditis.

Infective endocarditis (IE) - A disease caused by microbial infection of the endothelial

lining of intracardiac structures. Infections most commonly resides on one or more heart
valve leaflets but may involve mural endocardium, chordal structures, myocardium and
pericardium.

Native valve endocarditis (NVE) - Native valve endocarditis was defined as endocarditis
involving native heart valves and not prosthetic heart valves or implanted endovascular
devices. Patients with a pacemaker and/or implantable defibrillator could be included if
they had evidence of valvular infection and no evidence of lead infection. Injection drug
users were excluded because native valve endocarditis in this group of patients has
specific characteristics that differentiate it from native valve endocarditis in non-injection
drug users.

Prosthetic valve endocarditis (PVE) - A microbial infection of the endovascular that occurs
on parts of a prosthetic valve or on the reconstructed native valve of the heart. PVE
accounts for 20% of infective endocarditis. It is the most severe form of infective
endocarditis and is associated with high morbidity and mortality. Based on the time of the
disease acquirement, this disease is classified into two types, early and late PVE. The
early PVE is acquired within one year of the surgery, while the late PVE is acquired after
one year. The clinical importance of this classification are the distinct microbiological
profiles between the former and the latter.

Intravenous drug abuse (IVDA) endocarditis - Infective endocarditis (IE) is one of the most
severe complications in intravenous drug abusers (IVDA). IE usually involves the tricuspid
valve, Staphylococcus aureus is the most common etiologic agent, and it has a relatively
good prognosis. Currently, between 40% and 90% of IVDA with IE are HIV infected, and
the HIV epidemic has caused a decrease in the incidence of this disease, probably due to
changes in drug administration habits undertaken by addicts in order to avoid HIV
transmission.

MBBS Batch 27
List common organisms associated with infective endocarditis.

Streptococcus viridans
Pneumococci
Staphylococcus aureus
Enterococci
Coagulase-negative Staphylococci
Fastidious gram-negative coccobacilli
Gram-negative bacilli
Candida spp.
Polymicrobial/miscellaneous
Diphtheroid
Coxiella burnetii
Bartonella sp.

Explain ‘HACEK’ group of bacteria? Describe their common properties

and significance
HACEK is an acronym for a group of organisms that are small, fastidious gram-negative
bacilli:
H – Haemophilus
A – Actinobacillus
C – Cardiobacterium
E – Eikenella
K – Kingella

These organisms commonly colonize the human oropharynx as normal, indigenous flora
that could cause mouth infections. HACEK organisms are most often associated with
infective endocarditis, accounting for up to 10% of cases. Rarely, HACEK organisms may
cause severe systemic infections. Although most notable for causing infective
endocarditis, they are significant causes of other diseases including periodontitis,
abscesses, and septic arthritis. Baseline characteristics and predisposing factors of
HACEK endocarditis (HE) are, the median age of patients with HE was significantly lower
than non-HE and males predominated. Factors more commonly associated with HE than
non-HE endocarditis were Osler's nodes and vascular immunological phenomena and the
presence of mechanical valves. Factors less commonly associated with HE than non-HE
endocarditis were health care provision, and diabetes mellitus. There was no difference in
the proportion with fever or splenomegaly between HE and non-HE, nor with native valve
predisposition for IE or congenital heart disease.
CASE STUDY 1

A 50-year-old man with persistent fever and night sweats for the past 4
weeks suddenly developed increasing fatigue and shortness of breath. The
patient had a history of rheumatic fever when he was 15 years old. Eight
weeks back he had dental extraction without any antibiotic coverage.
On examination, he was febrile. His spleen was palpable. Splinter
haemorrhage and Osler’s node were detected in the right thumb and right toe
respectively. In echocardiogram, Tricuspid vegetation was detected. Blood
samples were sent for culture and sensitivity test.

1. Blood Culture- Green (alpha) hemolysis on blood agar

a. Gram-stain of the isolated organism - Gram-positive cocci in chains
b. Biochemical test- Catalase - ve, Optochin resistant and bile insoluble
c. ABST- Sensitive (S): Amoxycillin, vancomycin, Gentamicin
Resistant (R): Penicillin

Identify the causative organism (Explain the rationale for the diagnosis)

The causative organism is Streptococcus viridans. The organism is a Streptococci sp. because gram-
stain shows purple organism in chains presenting gram positive cocci in chains. No formation of
bubbles was observed from the catalase test confirming it as catalase negative. It is confirmed
Streptococcus viridian because green haemolysis was observed on blood agar and it is Optochin
resistant and bile insoluble.
State the indications for performing a blood culture? (How all
will blood culture help in the diagnosis?). Describe the
procedure of collection of blood sample (adult and children)
for culture.
A blood culture is a microbiological culture of a peripheral blood sample. As blood is
usually a sterile environment, culturing can show the presence of a systemic infection,
such as septicaemia. If the culture is positive, the causative micro-organism can usually
be identified, and antibiotic sensitivity testing performed. Endocarditis is an endovascular
infection associated with the persistent presence of infecting microorganisms in blood.
For this reason, blood cultures are the standard test to determine the microbiologic
aetiology of infective endocarditis. Routine blood cultures incubated on modern
automated, continuous-monitoring blood culture systems allow recovery of almost all
easily cultivable agents of endocarditis without additional specialized testing, such as
prolonged incubation or terminal subculture.

1. Adults: suspected bacteremia – draw two sets of blood culture bottles (aerobic and
anaerobic) from two separate venipuncture sites. Maximum of 4 cultures. The
aerobic bottle should be inoculated first as there is about 0.5 cc of air in the line of
the collection set and sometimes it is difficult to obtain 8-10 cc of blood per bottle
(15-20 cc/set). The aerobic bottle is the more critical one to inoculate short samples
into. The small lines on the edge of the label indicate approximately 5 cc and there
is a fill line denoted on the label. It is important not to underfill or overfill the
bottles as this can adversely affect the results. Pediatrics/Neonatal Patient: one
aerobic (yellow top) bottle as ordered by the physician. Recommended volume: 1
to 2 cc. of blood per bottle.
2. Blood Culture for TB: two heparin (green top) or two SPS (yellow top) Vacutainers.
No more than once per 24-hour period.
3. Blood Culture for Fungus: One aerobic (blue top) blood culture bottle.

Procedure
1. Locate the vein to be used.
2. Remove Frepp™ from package. Hold the applicator with ChloraPrep One-Step
Frepp™ sponge facing downwards and gently squeeze wings, releasing solution for
a controlled flow.
3. Place sponge on selected venipuncture site and depress once or twice to saturate
sponge.
4. Use a back-and-forth friction scrub for at least 30 seconds.
5. Allow area prepared to dry for approximately 30 seconds.
6. Proceed with collection of blood.

MBBS Batch 27
CASE STUDY 2
A 40-year-old male was admitted to the hospital with extensive third-degree
burns. Three days later, he spiked a fever, and there was pus oozing from the
skin was greenish in colour.
On examination, he had a temperature of 39.5⁰C, tachypnea (R>26/min) and
tachycardia (P>98/min) that suggested systemic inflammatory response
syndrome (SIRS)

1. Gram stain of pus: Shows gram negative rods and pus cells.
2. Blood Culture- Blue-green pigment on nutrient agar. Non-lactose-
fermenting colonies on MacConkey’s agar
a. Gram-stain of the isolated organism - Gram-negative rods
b. Biochemical test- Oxidase-positive
c. ABST- Sensitive (S): piperacillin, piperacillin+ tazobactam,
Ticarcillin+
tazobactam, Imipenem
Resistant (R): gentamicin, amikacin, ciprofloxacin
Identify the causative organism (Explain the rationale for the diagnosis)
Firstly, a sample of the pus is taken and a blood sample as well. Then, gram stain of the
pus is done. The result shows, gram negative rods and pus cells; which means it is a
bacillus. A gram-stained smear of isolated organism shows a clear view of Gram-negative
rods which indicates a pure culture. Hence, a biochemical test is done to confirm bacilli;
oxidase test. The test is positive since there is a change in colour to purple. This indicates
presence of bacilli. The culture medium used is nutrient agar and MacConkey’s agar. In the
nutrient agar, a blue green pigment is shown which proves Pseudomonas present and, in
the MacConkey’s, agar shows a clear yellow reagent, meaning, a non-lactose fermenting
colony. Based on these tests, it shows that is it Pseudomonas aeruginosa
Describe the major ‘blood culture criteria’ for infective
endocarditis (IE)
Typical microorganism for infective endocarditis from 2 separate blood cultures.

1. Viridans streptococci, Streptococcus gallolyticus, HACEK group.

Staphylococcus aureus

2. Community-acquired enterococci in the absence of a primary focus.

Persistently positive blood culture, defined as recovery of a microorganisms

consistent with infective endocarditis from:

- Blood cultures drawn > 12 hours apart

- All 3 or a majority of > 4 separate blood cultures, with first and last drawn at

least 1 hour apart

- Single positive blood culture for Coxiella burnetii or phase I IgG antibody titter

of >1:800