NAME

SCHOOL OF BIOSCIENCES

BI505 INFECTION AND IMMUNITY MODULE

MEDICAL MICROBIOLOGY PRACTICAL 2017

**IMPORTANT SAFETY INFORMATION **

IT IS ESSENTIAL THAT YOU REMEMBER AT ALL TIMES THAT ALL

MICROBES CAN BE POTENTIAL PATHOGENS. IN ORDER TO MINIMISE
POTENTIAL HAZARDS PROTECTIVE COATS MUST BE CORRECTLY
WORN WHILST IN THE LABORATORY. DO NOT EAT OR PUT PENS AND
PENCILS IN YOUR MOUTH AND WASH YOUR HANDS WHEN LEAVING
THE LABORATORY. IF THERE ARE ANY SPILLAGES PLEASE INFORM
THE LABORATORY SUPERVISOR, OR A DEMONSTRATOR,
IMMEDIATELY.REMOVE GLOVES BEFORE LEAVING THE LABORATORY

Introduction

This is a practical designed to enhance your understanding of disease

investigation, epidemiology, and infection control and prevention.

In this practical you will be working on a case study where you need to identify
the micro-organisms involved by analysis of the symptoms presented by the
patient, your use of diagnostic tests and stains on organisms isolated from the
patient and by the use of microscopy and text book information. You will also
interpret the antibiotic susceptibility of the organisms isolated from the patient

1
and personnel on the ward to help you to decide on the best course of action
in terms of treatments(s) after you have made a diagnosis of disease.

In order to guide you in this case study practical, specific comprehension

questions have been set. This is in addition to the experimental data you
obtain, e.g. the identification of organisms isolated from the patients and ward
personnel using diagnostic tests and the Table of the antibiogram results
obtained during the case study investigation.

Marks will be awarded for providing full experimental data/identification of

organisms and for answering the questions as comprehensively as possible;
the marks allocated to each question are indicated in brackets.

The deadline to submit your practical write-up online via the Moodle BI505 is
Wednesday the 12th April 2017 at 12 noon. Late work submitted past the
deadline of 12 noon will receive a zero mark, unless concessionary evidence
is provided, sent together with a completed online Biosciences concessions
form.

Case Study: A very young baby has been admitted to hospital

with blisters all over his upper trunk and face.

In this case, the medical officer of a local hospital has to identify (a) what is
the organism responsible and (b) who passed on the infection to the baby.

A baby of a few weeks of age has been admitted into hospital with blisters all
over the upper trunk and face. The attending Medical Officer takes swabs
from the skin itself and the skin lesions (blisters) and cultures the pure isolates
of the micro-organisms taken from the skin. They are plated onto

(a) blood agar (plates labelled isolate 1) (b) YEPD plates (plates labelled
isolate 2) and (c) two different organisms were plated onto MacConkey plates
(plates labelled isolate 3 and isolate 4).

Using the flow charts at the back of this manual, please identify the four
organisms that were isolated from the skin/blisters on the upper trunk of the
baby.

2
Start by doing gram stains (on microscope slides) on all four organisms using
the method provided in the Appendix, then look at the size and
shape/morphology of the colonies by eye and then look at the individual
organisms at different magnifications under the microscope. Do the organisms
have any smell? Once you have decided whether each microorganism is (a) a
gram negative bacteria (b) a gram positive bacteria or (c) another organism,
do the relevant diagnostic tests on the microorganisms with the help of the
flow charts to help you formally identify each organism.

1. What is organism 1 and what tests/observations did you carry out to identify
this organism? (10 marks)

2. What is organism 2 and what tests/observations did you carry out to identify
this organism? (10 marks)

3. What is organism 3 and what tests/observations did you carry out to identify
this organism? (10 marks)

4. What is organism 4 and what tests/observations did you carry out to identify
this organism? (10 marks)

The Medical officer decides that none of these organisms could have caused
the blistering on the babys skin so she then decides to take swabs from other
sites on the body away from the blisters on the trunk and face.

5. At which sites would you swab the body of the baby to get a sample of the
suspected pathogen for analysis? (4 marks)

6. This is a well-documented infection:

(a) What is the common name for this disease? (3 marks)

3
(b) What is the infectious agent affecting this patient? (3 marks)

(c) Why was this pathogen not found from swabs taken from skin blisters on
the baby? (3 marks)

(d) What does this organism produce that causes blistering on the skin? (3
marks) (12 marks in total for Q6)

In order to find the source of this infection, i.e. where the infective agent came
from, the medical officer then takes swabs from ward staff and patients and
isolates strains of Staphylococcus bacteria from all the nurses, doctors,
consultant and the baby (which are Patient 1 and Patient 2) present on the
ward.

These organisms (from the baby, the nurses, the doctors and the consultant
on the ward) were then analysed for antibiotic resistance/sensitivity using a
micro-broth dilution method. This allows the medical microbiologist to
determine the Minimum inhibitory concentration (MIC) of antibiotic which
stops the bacteria from growing (See Table 1). Table 1 shows which six
antibiotics were used AND what values of inhibition equate to either antibiotic
resistance or sensitivity for any tested bacterial species.

The results a) tell the microbiologist which antibiotic is likely to be an effective

antibiotic therapy to treat patient 1 and patient 2 and b) can be used by him
for epidemiological purposes by comparing the antibiotic resistance results
from the MIC tests from a range of suspected carriers on the ward with the
MIC results of ward staff and other patients, to see who was probably
responsible for infecting the baby (Patient 1).

Look carefully at Table 2, to see if the isolates found on the ward staff, the
baby (Patient 1) and a consultant on the ward (Patient 2) are either sensitive
or resistant to particular antibiotics. The consultant is called patient 2 and he
has an infected finger and has been seen by his GP. Table 2 also shows the
results of a Protein A test done on the isolated organisms from each tested
individual. Protein A is only produced by Staphylococcus aureus.

4
7. Record your results in Table 3 and present the completed Table 3 results
(including the identity of the staphylococcus isolated by filling in the end
column of the Table) as part of your final practical report.

(10 marks for a complete and correct Table).

The MIC (Minimum Inhibitory Concentration, in g/ml) for the six antibiotics
chosen was determined for each bacterial isolate. You are supplied with
these values and the EUCAST guidelines (See Table 1) used for
determination of resistance or susceptibility to the antibiotic. Using these
guide values, interpret the MIC for each of the values in Table 2. (The MIC for
each isolate was determined by making doubling dilutions of each antibiotic,
adding the bacteria and then allowing these bacteria to incubate in a 96 well
plate. The presence of bacterial growth at each concentration of antibiotic was
then detected by a Beckman Coulter walkaway 96 system device.
Table 1
MIC Range
Antimicr Sensi Resist
obial tive ant
0.12
Penicillin 5 0.125
Erythro
mycin 1 2
Fusidic
acid 1 1
Gentami
cin 1 1
Cefoxitin 4 4
Vancomy
cin 2 2

Table 2

5
The Protein A-based test results are already filled in for you (column 1). The
details of the test used are shown on page 7 of this manual. Please note:
Cefoxitin is a substitute for the methicillin antibiotic.

Write S for Sensitive and R for Resistant for each antibiotic in the boxes in
Table 2 above and then, based on the protein A and Cefoxitin information,
write in the type of Staphylococcus identified (in the last column). (This Staph
type could be CNS, Staph aureus, or MRSA). Transcribe this information into
Table 3 on the following page for inclusion into your practical report.

8. Have you been able to identify the carrier of the organism that infected the
baby? (This is based on the data in Table 3). (4 marks)

9. Assuming that you have identified the carrier, how would you treat the baby
and the adult carrier? For example, what antibiotic would be the best AND
also the least toxic to give to each, and would it be given in tablet form or via a
different route? (10 marks)

10. Is there likely to be a problem of poor infection control in the ward? Has
there been identification of a number of MRSA carriers within the ward staff,
based on your antibiogram results? If so, who are they and what is the best
way to treat such MRSA carriers? (10 marks)

11. To fully confirm that it was the carriers organism that was passed to the
baby, and thus was the source of the infection, what other, more definitive
diagnostic tests would you suggest should be done on both organisms to
100% confirm your suspicions? (10 marks)

6
 Table 3 Antibiotic Screening Results

Protein Penicil Erythrom Fusidic Gentamic Cefoxi Vancom Organism

A lin ycin acid in tin ycin ID
Nurse
1 Positive
Nurse Negativ
2 e
Nurse
3 Positive
Nurse
4 Positive
Doctor
1 Positive
Doctor Negativ
2 e
Patient
1 Positive
Patient
2 Positive

Write S for Sensitive or R for Resistant for each antibiotic in the boxes in
Table 3 above and then, based on the protein A and Cefoxitin information,
write in the type of Staphylococcus identified based on this information (in the
last column). This Staph type could be coagulase-negative staph (CNS),
Staph. aureus, or MRSA.

7
 APPENDIX

The following information outlines the methods you can use today to
identify the organisms provided in this practical. These methods should be
used in conjunction with the two flow charts at the back of this Appendix.

GRAMS STAIN METHOD

Place a large drop of water on the slide.

With a plastic loop emulsify a colony of the organism in the water, by mixing and
stirring.

Heat fix by briefly drying over a Bunsen flame. approx. 3-5 sec. (Do not overheat
or you will destroy the morphology of the microbe).

Add a few drops of Crystal violet to cover the slide and leave for approx. 5 sec.

Wash in tap water and allow excess fluid to drain from the slide.

Add a few drops of Lugols or Grams Iodine to just cover the slide and then leave
for approx.10 sec.

Decolourise with Acetone. This reaction should be instant.

*IMPORTANT SAFETY NOTE: KEEP BUNSEN BURNER AWAY FROM THE

ACETONE: THE VAPOUR IS HIGHLY INFLAMMABLE!)*.

Wash in tap water and allow excess fluid to drain from the slide.

Counter stain with Safranin or Neutral red approx. 5 sec.

8
 Wash in tap water.

Blot dry, using paper towel, and examine using x10 objective and then x 100 oil
objective on the microscope.

Gram positive organisms will look dark purple and Gram negative organisms will
appear pale pink under the microscope

PLEASE CLEAN THE 100 OBJECTIVE LENS WITH ALCOHOL/IMS (AFTER

WIPING OFF EXCESS OIL WITH LENS TISSUE) AND THEN DRY THE
OBJECTIVE LENS WITH LENS TISSUE AFTER YOU HAVE FINISHED
LOOKING AT THE SLIDE WITH THE USE OF IMMERSION OIL.

PLEASE CLEAN THE MICROSCOPE STAGE AS WELL TO REMOVE ANY

IMMERSION OIL.

STAPH AUREUS (PROLEX) LATEX KIT

Intended Use:

This is one of a series of commercial products designed to produce a rapid slide

agglutination test to confirm whether Staphylococcus aureus is present. It will
differentiate those staphylococci that produce the enzyme coagulase and/or protein A
(particular protein layer in the cell wall of Staphylococcus aureus). This test should
differentiate the pathogen Staphylococcus aureus, from staphylococci such as
Staphylococcus saprophyticus which possess neither of these factors, which are
invariably non-pathogenic.

9
It has been demonstrated that Staph. aureus cultures possessing clumping factor (an
exo protein that has the ability to clump plasma) and protein A (a particular component
in the cell wall of Staph. aureus) can be identified using human plasma-coated latex
particles, which agglutinate in a rapid slide procedure. The Staph. aureus latex reagent
consists of latex particles, which have been coated with fibrinogen and IgG. When
mixed on a slide with a suspension of Staph aureus organisms, the reaction of
clumping factor with the fibrinogen, and/or of protein A with the IgG causes rapid,
strong agglutination of the latex particles.

Test Procedure:

1. Shake the latex to obtain an even suspension and dispense a small drop into a
circle on the reaction card for the particular culture to be tested.

2. Take a plastic loop and pick up some of the culture (2-3mm diameter) by touching it
with the end of the loop. As a guide, an amount of growth roughly equivalent to six
average-sized colonies should be picked.

3. Emulsify the sample of culture by mixing it (using a wooden stick) into the drop of
latex suspension on the card. Rub thoroughly but not too vigorously as the surface
of the card may be damaged. Spread the latex suspension over the whole area of
the circle. Discard the plastic loop and the wooden stick into a biohazard bag for
safe disposal.

4. Rotate the card gently and examine for agglutination for approximately 20 seconds,
holding the card at normal reading distance (25-35cm) from the eyes.

5. Do not throw the card into a biohazard bag as others can use the spare spaces.

Reading of Results:

A positive result is indicated by the development of an agglutinated pattern showing

clearly visible clumping of the latex particles with clearing of the milky background.
Most positive reactions will be almost instantaneous.

10
A negative result is indicated when the latex does not agglutinate and the milky
appearance remains substantially unchanged throughout the test. It should also be
noted that traces of granularity might be seen in negative patterns due to the
particulate nature of both reactants.

11
 CATALASE TEST

BACKGROUND INFORMATION

A number of organisms such as staphylococcus contain an enzyme called catalase.

This enzyme is a natural defence mechanism which breaks down super-oxides which
build up inside cells. This can easily be detected using hydrogen peroxide.

SAFETY NOTE:

This test should never be done on a glass side as the bubbling produced causes
aerosols. It must be carried out in an LP4 capped tube as the walls of the tube
retain most of the splashing.

1. Locate your small (LP4) plastic test tube on your bench which contains 0.5ml
hydrogen peroxide.

2. Lift one of the colonies you wish to test with the end of a plastic loop and put onto
the inside wall of the tube. Cap the tube and rotate the tube until the hydrogen
peroxide covers the organism.

3. A positive reaction is indicated by the rapid effervescence (bubbles) from the colony,
indicating that the catalase enzyme present in the bacteria sample is liberating
oxygen from the hydrogen peroxide.

12
 FERMENTATION OF LACTOSE TEST

The ability of organisms to breakdown lactose to lactic acid is one of the simplest
microbiology tests.

MacConkey agar contains lactose, bile salts and phenyl red as its reactive
constituents. The bile salts only allow the growth of bile-tolerant organisms (those that
live in the gut); the lactose is broken down to lactic acid, which turns the phenyl red
indicator from colourless to red. Thus lactose-fermenting colonies grown on this agar
are red and non lactose-fermenting colonies are colourless.

13
 FLOW CHART FOR ORGANISM IDENTIFICATION

(A) GRAM NEGATIVE ORGANISMS

COLOUR CHANGE PRESENT ON BACTERIAL GROWN ON YEPD

ISOLATES GROWN ON MAC CONKEY AGAR? AGAR? LARGE ORGANISM?

SMELL OF BREAD?

NO? YES?

Saccharomyces

cerevisiae

NON-LACTOSE

FERMENTERS LACTOSE FERMENTERS

14
Proteus vulgaris Escherichia coli

15
FLOW CHART FOR ORGANISM IDENTIFICATION

(B) GRAM POSITIVE ORGANISMS

COCCI SHAPE ROD SHAPE

AEROBIC ANAEROBIC

Corynebacterium spp. Clostridium spp.

CATALASE TEST

16
 POS NEG

Staphylococci Streptococci

COAGULASE TEST -Haemolysis on blood agar?

Positive Negative Positive Negative

S.aureus S. saprophyticus Haemolytic Strep. Non-Haemolytic Strep.

17
 Practical Write-up Information

Please submit your write-up online on the relevant part of the BI505
Moodle page (practical submission). The deadline is 12 noon on
Wednesday the 12th April 2017, but you can submit before this deadline
if you wish. The practical will run twice, with half the class attending at
each session on the 31st March (10-1 or 2-5). Both practical sessions
will use the same manual, but not necessarily the same organisms.

This practical is part of your continuous assessment for this module.

High marks will be awarded for correct, fully completed answers, which
demonstrate both a clear understanding of the practical, and clarity of
presentation.

You are welcome to bring your notes and textbooks (such as Mims
Medical Microbiology) into the practical, to aid your diagnoses. Your
practical write-ups will be marked using a designated and detailed
marking scheme and we will return it to you with feedback in the
summer term.

18
COSHH Regulations 1988. This manual has been subjected to a formal
risk assessment. It advises students of the risks arising from their work
and of the precautions to be taken.

Signed: Lis Curling Date: March 2017

Dr Elisabeth Curling, Module Convenor

19