2017

August

MICROBIOLOGY LAB REPORT 1

Prepared By: Samira Abdullah MD0166/B3/08
Introduction:
Lab safety rules are useful for many reasons. The laboratory is a
place where all kinds of potentially disease causing pathogens are
processed. A person working in the lab may unknowingly infect himself or
others who are in contact with him. Not only that but he may contaminate
the specimen he’s working on and render false positive and false negative
results. He might injure himself or his colleges mechanically or chemically.
All the above happen if lab rules aren’t followed.

Lab safety rules:

1. Wear clean gowns, buttoned up to the neck, and leave the gown in the lab after you
finish working. This is to reduce spread of microbes.
2. Wear gloves, eye goggles, complete appropriate footwear, no loose clothing and long
hair must be tied to the back to avoid catching fire. All personal protective equipments
should be used. Show no skin.
3. Follow the ABC rule: ALWAYS BE CAREFUL.
4. Keep your pathway clear by placing extra items like books, bags etc. on shelves or under
the tables so they won’t be stepped on.
5. Wash your hands before and after performing procedures.
6. Don’t taste or smell chemicals as they are injuries to your taste buds and olfactory cells
respectively. Don’t put anything in your mouth, not even your pen.
7. Don’t store food in the lab fridge.
8. Turn off Bunsen burner after use.
9. Safety box should be used for broken glass and sharp materials.
10. Treat everything in the lab as infectious so as to avoid mistaken contamination.
11. Don’t attempt unauthorized procedures.
12. Label your specimen as quick as possible to avoid pre-analytical error. Avoid analytical
error and post analytical error.
13. Be very careful while using glass wares so as to avoid their breakage and spilling of
chemicals on you.
14. Report all accidents, injuries and breakage of glassware to the instructor immediately.
15. Location of fire extinguisher, eye wash station, first aid kit and safety shower should be
known as optimal requirement at the first laboratory visit. Used in case of emergency.
16. Don’t lean, hang over or sit on the laboratory tables.
17. Follow all the instructions given by the instructor.
18. Needs and inoculation loops should be sterilized to have a reliable result.
19. Keep our work area aseptic (before and after procedure) and your techniques too.
 20. Don’t eat, chew gum or drink in the laboratory.
21. Clean and keep your work station ordered before you leave the lab.
22. After using the microscope, rotate the focus to 4X, lower the stage, turn off the light,
unplug it from electrical source and cover it with the custom made plastic bag.
23. Know the meaning of the hazard signs. Here are some of them:

List of equipments found in the lab:

1. Autoclave  used to sterilize “thermo stable” culture media, glassware etc.
2. Incubator  provides microorganism optimum temperature to grow in vitro.
3. Hot air oven  similar to incubator but (3000C) to sterilize thermo unstable equipments.
4. Inoculating loop  tool for transferring and streaking cultures.
5. Straight wire  to inoculate specimen into a test tube.
6. Pipette  lab tools used for measuring and transferring different chemical and
organism solutions.
7. Water bath  for heating and melting of media, solutions, etc. at temperature below 100 0C.
8. Vortex mixer  used to mix liquid in test tube by shaking the test tube.
9. Hotplate with magnetic stirrer  heats and agitates using magnetic beads.
10. Inoculation chamber  used for highly infecting disease like Ebola, Yersinia Pestis etc.
11. pH meter  used to maintain pH of medium and diluents.
12. Microscope  used to observe microorganism at a higher level of magnification.
13. Refrigerator  to store/preserve culture media, and many sensitive materials.
14. Bunsen burner  to sterilize inoculating loop, create sterile zone for aseptic operation.
15. Spirit lamp  has similar function as Bunsen burner but is portable.
16. Caliper  to check the length of susceptibility of a pathogen for
a drug.
17. Digital balance  to weigh chemicals, samples, media, etc.
18. Thermometer to ensure heating equipment runs at correct
temperature.
2017
19. Reagent bottle  used to keep small amounts of solution
rather than the stock.
20. Centrifuge used for separating substances by their weight.
21. Test tube rack to keep test tubes in place.

Glasswares:
22. Beaker  used for mixing, measuring and temporary storage of reagents.
23. Flask used for mixing chemicals mainly.
24. Test tubes used for biochemical testing and mixing chemicals in small amounts.
25. Funnels  used to cleanly add liquid material from one bottle to the other in clean
manner.

Chemicals:
26. Agars: Mac Conkey agar, blood agar etc.  used to grow microbes on growth media.
27. Alcohols: Ethanol, Methanol, Iodine, Safranin etc  used for staining microbes.
Session 2

Gram staining:
It is named after a Danish bacteriologist Hans Christian Gram who developed the technique. It’s
a staining method used to differentiate between two diverse categories of bacteria based on
their (chemical and physical) cell wall structure. The gram positive and gram negative bacteria
are differentiated that enable us to further classify the bacteria species. Cocci, Bacilli and
spirochetes are the different types of bacteria that exist.

Materials:
1) Microscope slide
2) Inoculation loop
3) Physiological saline
4) Wax pencil
5) Bunsen burner
6) Staining rank
7) Pippete
8) Gauze
9) Microscope
10) Immersion oil
11) Distilled water
12) Sample
13) Reagents placed in test tubes:
a. Crystal violet
b. Iodine
c. Acetone/ Ethyl Alcohol
d. Safranin

Preparing heat fixed smear:

1) Place a drop of normal saline on the slide
2) Obtain a small amount of specimen, using an inoculating loop and place it on the saline
and mix it thoroughly. Be careful not to add too much or too little specimen.
3) Air dry the sample.
 4) Now heat the slide using a Bunsen burner/ spirit lamp by moving the slide on top of it.
Move it right and left for 4-5 times to make it fixed on the slide. Fixing helps the bacteria
not to rinse during further procedures.
5) Your slide is ready for further processes.

Precautions:
1) If the bacteria has no or a damaged cell wall it won’t stain.
2) Longer time stay of alcohol washes away the stain leading to False Negative.
3) Different composition of cell wall like lipid in M.tuberculae won’t stain.

Procedure:
1) Bring the heat fixed smear slide and place it on a staining rack.
2) Now using a pippete cover the slide with crystal violet. Wait for 1 minute. Crystal violet
dissociate into CV+ and CL- ions these ions enter the cell wall and CV+ interacts with
negative charged components of both gram positive and negative and stains them.
3) Now rinse the slide with distilled water and add iodine. Wait for 1 minute again. The
iodine interacts with the CV+ that has remained in the cell wall and makes a complex
keeping or retaining the stain.
4) Rinse the slide with distilled water and add acetone for 5-10 seconds only. To avoid the
washing of the whole smear don’t make it stay longer. Acetone removes the stain
complex from thin cell walled bacteria (negative) and dehydrates the complex in the
thick cell walled bacteria (positive). Hence retaining its staining property by crystal
violet.
5) Now cover the slide using the pippete by Safranin and wait for 45 seconds. The thin cell
walled bacteria will stain by it whilst the thick cell walled bacteria won’t.
6) Rinse the slide with distilled water again and wipe the under part of the slide by gauze.
7) Let it air dry, apply oil immersion and observe it under the microscope by x100.

Observation:
The bacteria that appears blue to purple (Crystal violet) is gram positive whilst that appears
pink to red (Safranin) is gram negative. Observe the morphology of the bacteria, presence
or absence of pus cell, epitheliod cell and yeast cells (as they retain the bacterial counter
stain)
Report:
- Gram Positive Bacilli
- Gram negative bacilli
- Weakly gram positive cocci
- Gram positive Staphylococcus
- Gram Positive Streptobacilli

Conclusion:
Since bacteria’s differ in their cell wall we are able to differentiate them in to
gram positive and negative by using the gram stain procedure. This helps us in
clinical medicine because the antibiotics given for the two differ.
 Session 3:

Acid fast stain:

First described by two German doctors Franz Ziehl and Friedrich Neelson and hence named
after them. It is used to identify Mycobacteria species and a few other bacteria species.
Mycobacterium tuberculea is the most important of its species as it causes tuberculosis.
Acid fast organisms contain large amount of lipid in their cell wall called mycolic acids which
resist staining by gram stain; hence, the use of acid fast stain on them. They appear bright
red after staining if they are acid fast organisms.

Materials:
1) Zieehl’s Carbol Fuchsin (10 Stain)
2) Acid Alcohol
3) Methylene Blue (counter stain)
4) Clean microscope slide
5) Bunsen burner
6) Staining rack pipette
7) Distilled water / tap water
8) Specimen (sputum)
9) Applicator
10) Immersion oil
11) Microscope

Regarding the specimen:

Sputum is used to diagnose Pneumonia, Tuberculosis, Bronchitis, etc. criteria for sputum
collection:

1) Should be coughed from the lungs and not from throat. (Quality)
2) Should be mucus and not saliva. (Quality)
3) If TB is suspected 3 sputum samples. Taken at spot-morning- spot. Morning is
preferred. Take different specimen in different cup.
4) 15 ml is sufficient quantity.
5) Be sure to have a labeled, good quantity and quality, fresh and non-contaminated
specimen.
6) Collect the specimen using a cup or nasotracheal suctioning.
Procedure:
1) Prepare a heat fixed smear as discussed previously.
2) Now place the heat fixed slide on a staining rank. Flood the slide with Carbol Fuchsin
using a pipette and heat it using a Bunsen burner whilst it’s on the rank until flames
are seen. Heat is used so that the stain will enter the thick waxy lipid layer of the
acid fast bacilli. Wait for 3-5 minutes. Rinse the slide with tap water.
3) Now again place the slide on the staining rank and flood it with methanol. The acid
washes away the stain from the non-acid fast bacilli as they don’t have thick waxy
mycolic acid cell wall. Wait for 20- 30 seconds. Rinse with tap water
4) Flood the slide with Methylene blue/Malachite Green (counter stain) and wait for 2-
3 minutes. This stains the other cells that were washed off the primary stain. Wash
with water clean the down side of the slide and leave it to dry.
5) Apply oil immersion and observe under the microscope at X100.

Observation:
1) If the bacilli appear reddish/ pinkish (retain Carbol Fuchsin), straight or slightly curved 
acid fast bacilli
2) They will be backgrounded by cells colored blue(in case of methylene blue) or green (in
case of malachite green)

3) Observation under 1 field

 if >10 AFB then report +++
 If 1-10 AFB then report ++
4) Observation under 100 fields
 If 10-100 AFB / field then report +
 If 1-9 AFB/ field then report exact number.

Report:
The observed slides were:
1) AFB/field _ _ _ _ _ _ +++ (>10 AFB/field)
 They showed more than 10 bacilli.

2) AFB/field _ _ _ _ _ _ ++ (1-10 AFB/ field)

Another showed less than ten;

3) After staining the given sample, there were no acid-fast bacteria observed.
Session 4:

Preparing culture media:

Media are used to isolate and identify bacteria, reveal their metabolic properties, and allow
long-term storage of pure cultures. The study of microorganisms requires techniques for
isolating cells from natural sources and growing them in the laboratory. Culture media are
prepared in order to make the environment suitable for bacteria to grow in vitro. Providing the
suitable nutrients, temperature, and essential requirements will allow the proper growth of
bacteria. Not only this, but several other culture media serve as test bases for properties of the
bacteria, in order to place them in their respective species. Thus, culture media and culture
techniques have played important roles in the advancement of this field.

 TYPES OF CULTURE MEDIA:

A) Physical classification:
1) Solid Agar
2) Semisolid Agar
3) Liquid Agar
B) Chemical Classification:
a. Synthetic
b. Complex
C) Functional Classification:
a. Complex media: prepared media by boiling animal or plant materials to
extract nutritive molecules. Contents like peptone, meat extract, yeast,
carbohydrates, etc
b. Defined media: formulated from pure substances at predetermined
concentrations.
c. Selective media: they support the growth of only certain types of bacteria.
d. Differential media: reveals specific metabolic or metabolic characteristics of
bacteria grown on it.
Basic classes of culture media:
1. Basic/ simple/ all purpose media
2. Enriched/ solid media
3. Blood agar
4. Enrichment media
5. Transfer media
6. MacConkey agar
  Agar= an inert polysaccharide extract of the plants which is used to solidify culture
media and isn’t metabolized by the bacteria.

Materials:
1) MacConkey Agar
2) Flask (Sterile)
3) Measuring cylinder
4) Analytical balance (paper to place the powder on)
5) Hot plate
6) Distilled water
7) Petri dishes (Sterile)
8) Incubator
9) Autoclave
10) Spirit lamp
11) Aluminum foil
12) Sterile gauze
13) Refrigerator

Procedure:
1) Bring the MacConkey agar container and measure 8 gram of the powder on the
analytical balance. To make things easier first place a paper (1.1 gram) over the balance
and zero out the weight of the paper.
2) Measure 200 milliters of water and pour it to a flask.
3) Now add the powder to the flask and move it in a circular manner to mix the powder
with the water. Cover it with aluminum foil to avoid contamination.
4) Place the flask on a hot plate and turn it on. Make sure to mix the contents of the flask
by moving it in a circular manner every once in a while. Wait until the solution starts to
boil and then remove the flask. Pick the flask with sterile gauze to not burn your hands.
5) After making sure the contents are mixed thoroughly, autoclave the flask at 120 0C for
15mins.
6) Let the flask and its contents cool down for a short while.
7) Now pour the contents into the sterile petri dishes. Flame the mouth of the flask using a
spirit lamp before pouring and do not open the petri dishes more than the require
 amount to avoid contamination. The spirit lamp will also remove the airborne
microorganisms if kept on through the process.
8) Allow the solutions to cool down by opening the lids of the petri dishes slightly until
they solidify and then close them and take them towards the incubator and place them
inside the incubator which should be setup at 370C. Place the petri dishes at an upside-
down position in order to avoid the vapors collecting on the top roof of the petri
dishes.
9) Wait for 24 hours and take out the petri dishes.

Result:
1) Some Petri Dishes were clean and had no growth on them hence were put in the
refrigerator for future use.
2) Some of the petri dishes had bacterial growth on them hence were discarded.
Session 5:

Culturing bacteria and aseptic technique:

Bacteria are ubiquitous and are found everywhere growing on almost any substrate. Aseptic
techniques should be used in order to avoid unwanted growth of bacteria in the laboratory. The
laboratory accepts specimens to detect the presence of potential pathogens in the specimen of
a patient suffering from a certain disease. In the laboratory important component of culturing
bacteria involves isolating a single species from a mixed culture, where several species might be
present and identifying the specific pathogen. Moreover, care must be taken to provide as
direct method as possible when transferring a swab sample to media for culturing so as not to
introduce bacteria from unwanted sources. Slant tubes and agar medias are used to streak
with bacteria.

Inoculation = the transfer of a substance from one place to another.

Streaking = isolating a pure strain from a single species of microorganisms.

Butt and slant tubes = used to check fermenting ability of bacteria and gas production.

Material:
1) Petri Dish with required fresh culture media
2) Inoculating loop
3) Spirit lamp
4) Sterile swab
5) Sterile test tube
6) Vortex mixer
7) Incubator
8) Distilled water
9) Test tube rack
10) Urine sample
11) Marker

Procedure:
*** Before starting any procedure clean your working area thoroughly.***

A) For a fresh solid sample:

1) Take the test tube and put two to three drops of distilled water into it and place the
swab inside it (to moisten it). Cover the test tube with its cover.
2) Go out to search for the desired specimen (in this case lift handle and doorknob). Take
out the swab and gently rub over the surface (make sure not to include dust on the
swab). Place back the swab in the test tube mix it with the water found inside the test
tube. Cover the test tube ( don’t close it tightly to avoid death of strict aerobic bacteria)
and head back to the laboratory.
3) Place the test tube on the rack and wear gloves.
4) Bring up the required Petri Dish and label it. (You can divide the petri dish into 4 quarters
in order not to waste resources).
5) Bring the inoculating loop and flame sterilize it under the spirit lamp. Cool it in the air for
2 seconds or by placing it on the wall of the test tube for 1 second. Holding the
inoculating loop by your right hand, bring the test tube out of the rack using your left
hand. Open the cover using the medial surface of your right hand and insert it in the
specimen and take a sample. Cover the test tube and place it back to where it was.
6) Using your left hand open the Petri Dish and place it at your eye level and start streaking
in a zigzag fashion starting from the left top going right and low until you reach left
lower part. Turn it again and streak it in the same manner but at right angles to the first
streak. Perform the third and fourth streaks in the same manner. Zigzag manner
streaking is needed in order to spread growth of bacteria. After streaking flame sterilize
the loop and place it back.

7) Cover the petri dish and place it upside-down in the incubator at the required
temperature (in this case 370C. wait for 24 hours). CO2 should be provided for
capnophilics, using anaerobic jars for anaerobes.

B) For an old culture specimen:

1) All the above procedures are done except for the specimen being the isolated colony of
bacteria is taken as a specimen.
 C) For a urine sample:
1) Wear gloves, bring up the required Petri Dish and label it and divide it into two (using a
marker draw at its back).
2) Bring the urine sample container and take specimen via inoculating loop (after flame
sterilizing the inoculating loop).
3) Start streaking the petri dish in the same zigzag manner from top to bottom on the
whole petri dish. After streaking flame sterilize the loop and place it back.
4) Cover the petri dish and incubate at the required temperature and hours.

D) Inoculating a butt & slant test tube:

1) Bring a straight loop and insert it into the specimen.

2) Take the butt and slant test tube and stab the butt (make sure to not touch the sides
of the test tube) and immediately after removing the inoculating tube from the butt,
streak the slant in a zigzag fashion.

Result:
Check the following characteristics of the bacteria:

1) Shape: Circular, Rhizoid, Irregular, Filamentous or Spindle.

2) Elevation: Plain, Convex, Concave, or Raised.
3) Size: Small, Medium, or Large.
4) Texture: Smooth or Rough
5) Appearance: Mucoid
6) Pigmentation Purple, Yellow, or Creamy
7) Optical Density: Opaque, Translucent, or Transparent
For UTI testing:

Check the following to conclude results.

Loop capacity CFU/ml

0.001ml Colony 103

0.01ml Colony 102

105 CFU/ml; Bacteria is significant

105 CFU/ml; Bacteria is insignificant

Observation:
Session: 6

Coagulase and catalase test for gram positive bacteria

Coagulase is a protein enzyme produced by
many microorganisms and enables them to
convert fibrinogen to fibrin, hence enables them
to clot blood on their surface. This allows them
to survive the host defense system. Mainly
coagulase is used to identify Staphylococcus
aureus from the rest staphylococci as they are
coagulase negative. Whilst catalase is a protein
enzyme that reduces hydrogen peroxide to
oxygen and in some cases oxygen peroxide. It is
used by aerobic, facultative aerobes and micro-
aerophilic to avoid the oxygen radical damage
due to the use of aerobic respiratory pathway.
The inability of strict anaerobes to synthesize
catalase (peroxidase, or superoxide dismutase)
may explain why oxygen is poisonous to these
microorganisms and they can’t be cultured or
able to live in the presence of oxygen.

Both the above tests are used to distinguish different types of bacteria in the laboratory.

MATERIALS:
1) Microscopic slides
2) Culture containing cultivated specimen (bacteria)
3) 3% Hydrogen peroxide solution
4) Dropper
5) Normal saline
6) Tooth pick
7) Distilled water
8) Citrated plasma
Procedure:
A) CATALASE TEST (slide method):
1) Bring a clean slide and place it on sterilized table.
2) Place a small drop of normal saline in its center.
3) Take a tooth pick and pick up a small amount of culture from the petri dish and place it
on the drop. Be sure to pick up an isolated colony to avoid mixed bacteria. Avoid
accidentally taking blood Agar as it will result in false positive.
4) Emulsify the taken colony on the drop until homogenized.
5) Take a dropper and draw hydrogen peroxide solution from the container and place a
drop on the emulsified solution on the microscopic slide.
6) Observe the fluid for appearance of bubbles on it. Note your observation.
7) Now discard the slide into disinfectant filled jar with precaution.

*** Weak catalase or pseudocatalase reaction may be produced by some strains

of Aerococcus species and Enterococcus species. ***

OBSERVATION:
1) Gas bubbles were produced and the bacteria were identified a catalase positive bacteria
(staphylococci bacteria).

B) COAGULASE TEST (slide method):

1) Take a clean microscope slide and place it on a sterilized table.
2) Place a small drop of water in its center.
3) Bring a tooth pick and take colony sample from the already cultured petri dish.
4) Now emulsify the taken colony in the drop of water and homogenize it.
5) Now add a drop of citrated plasma (human plasma in this case) using a dropper and mix
it well using tooth pick.
6) Rotate the slide in a way that the mixed fluid rotates in a circular motion.
7) Coagulation will occur in a minimum of 5-10 seconds. Observe and note your
observation.

OBSERVATION:

1) The coagulation test had coagulation and the bacteria were identified as coagulase
positive (typical characteristics of S.Aureus).
Session: 7

Lab identification of gram negative bacteria

Gram negative bacteria require a series of biochemical tests to be differentiated to species
level. Especially after being cultured in differential media. MacConkey agar serves as a
differential and selective media. Selective: Gram positive is not allowed to grow on it.
Differential: Isolates the lactose fermenter from the non-lactose fermenters. As for the
biochemical tests some of them are listed as below:

1) Triple Sugar Iron Agar Test:

In this test three types of sugar (lactose, glucose, and sucrose) are arranged from top to
bottom in the test tube in a butt and slant method. Their color is primarily pink. After
stabbing and streaking the butt and slant if the color changes to yellow that means there is
fermentation of the specific sugar. Fermentation at top means lactose fermenter, at the
middle shows glucose fermenter, and at the bottom means sucrose fermenter. If the whole
column is pink that means the bacteria ferments all sugar types. Presence of crack and
uplifting of agar means there is production of gas. Mostly gas (CO2 and H2) formation is due
to glucose fermentation. The darkening of color towards black will indicate the production
of H2S. Darkening of the color is owed to the ferrous sulfate being converted to ferrous
sulfide and H2S gas. Phenol red and ferrous sulfate serves as indicators of acidification and
H2S formation, respectively.

2) Indole Test:

This test determines the ability of the organism to use tryptophan molecule into Indole.
If the bacteria has tryptophanase enzyme it can deaminate tryptophan and produce Indole,
pyuruvic acid and ammonia. Mainly used to differentiate the enterobacteriae family. We
use a tryptone broth (has tryptophan) and introduce the bacteria after incubation for a day
at 370C. Add 15 drops of Kovac’s reagent. The appearance of bright red color will indicate
the bacteria to be Indole positive.

3) Oxidase Test:

The purpose of this test is to check the ability of the bacteria to oxidize tetramethyl-p-
phenylenendiamine dihydrocholride to indophenol. This will lead us to the conclusion that
the bacteria have cytochrome oxidase. If there’s no change in color that means the bacteria
lacks the component.
4) Urease Test:

Test done to determine the ability of the bacteria to hydrolyze urea to ammonia using the
enzyme urease. This technique in the host body allows the bacteria to survive acidic
environment and neutralize the acid with alkaline. We use urea broth and the color is
yellow but when inoculated with the bacteria and after the action of its urease will be
converted to pink. Hence, proving the presence of the enzyme urease in the bacteria.

5) Simon Citrate Test:

This test is based on the ability of the bacteria to utilize citrate as its only source of carbon
and ammonia as its only source of nitrogen. Simmon Citrate Agar with a slant and butt is
used to perform this test. After the stab and streak of the butt and slant and incubation for
24 hours at 370C the color of the agar changes from green to blue. This change of color is
due to alkaline reaction following citrate utilization.

6) Motility Test:

The purpose of this test is to differentiate motile and non-motile species of the bacteria.
Media containing tryptose broth is used. The bacteria are stabbed at its center using
inoculating needle and growth is observed. If there’s growth away from the center of the
stabbing the test is positive and the bacteria is motile and vice versa.

OBSERVATION:
Under the microscope it was observed:

Slide 1) Gram positive bacilli (pale) and few gram negative

Slide 2) Gram positive cocci

Slide 3) Gram negative rod

Slide 4) S.pnemonae: gram positive diplococcic

Slide 5) Gram negative diplococcic found intracellular in pus cells

Session: 8

Urine Analysis and Blood Culturing

Blood sample collection is done in order to support clinical suspection and lead to a perfect
diagnosis. To take a blood sample we must consider the time and amount of blood taken from
the patients to avoid false results. Moreover avoiding contamination of the specimen and
taking it before administration of antibiotics are also crucial. The amount of blood taken for
adults is 20ml while for children just 5 ml is enough. It should be taken three times (every 8
hours) a day. Preferably, take at febrile time as that is the time of bacteria release and its
byproduct. We use blood culture bottle in order to grow the bacteria. This blood culture bottle
contains:

1) Thioglycolate broth (for anaerobic)

2) Tryptone soya broth (for aerobic)

After the inoculation of bacteria we observe the bottle for growth of bacteria which will give us
reason for the disease of the patient. Sub culturing, biochemical tests are done to identify the
specie of the bacteria. Antimicrobial tests, to detect resistance/susceptibility and give the right
type of drug to the patient.

Materials:

1) Tourniquet
2) Sterile syringe
3) Cotton dipped in 70% Ethanol
4) Blood culture bottle with the required broth.
5) Incubator

PROCEDURE:

1) Wear gloves and apply tourniquet on the patients arm at the two thirds of his arms. This
helps to engorge the vein so you’ll be able to easily localize it.
2) Now locate a vein on the arm and apply the cotton dipped in 70% ethanol on the area
around it. Be sure to rub from inside out (if available use 2% tinctures iodine too).
3) Now bring the sterile syringe (make sure the level is upward) and puncture the vein and
draw the required blood as mentioned above.
4) Release the tourniquet (to avoid oozing of the blood from the puncture) and pull the
syringe out.
5) Clean the bush of the blood culture bottle with 70% ethanol and stab it with the syringe
and inject the blood into the culture.
 6) Now incubate and observe the bottle for 3-7 days (for brucella 2 weeks). If there is
turbidity, hemolysis, and distinct bacterial growth indicate presence of bacteria.

B) URINE ANALYSIS:
Normally, urine in the bladder and the urinary tract from kidney to the last third of the urethra
is sterile. Urine analysis is used when urinary tract infection is suspected. Uropathogens like
uropathogenic E.coli, Chlamydia etc. are the culprits in this case. There could be upper UTI or
lower UTI based on the route of infection. The patient provides the urine sample in a specimen
cup that has wide neck, is sterile and doesn’t leak. Proper instructions should be given to
patient on providing mid-stream urine to avoid false positive due to the normal flora bacteria
present in the beginning stream of the urine and 20ml (half cup) on the average is needed.
Routine urine analysis includes:

1) Physical Examination
2) Chemical examination
3) Microscopic examination

The physical examinations include turbidity, color, volume, etc. the chemical examination
includes the dip stick method which tests for proteins and other components that shouldn’t be
found in the urine or for instance its ph. Microscopic examination includes the centrifugation of
the urine sample in a test tube and centrifuging it to yield sediment and supernatant. The
supernatant is removed and sediment is further examined under the microscope. Examination
of urine sediment may reveal the presence of different types of cells such as epithelial cells,
leukocytes, erythrocytes, or renal cells. Different types of crystals, yeast, bacteria, or casts may
also be present. Microbiological examination could also be carried on by using urine culture.
Urine culture is performed in the same way the blood culture is. The only difference is the
inoculating tube is replaced by calibrated loop and the culture by either MacConkey or CLED
(cystine lactose electrolyte deficient). The whole petri dish should be used for urine culturing.

MATERIALS:
1) Urine cup
2) Urine analysis strips
3) Centrifuge
4) Test tube and test tube rack
5) Microscope slide and cover
 6) Microscope
7) Gloves

DIP STICK PROCEDURE:

PROCEDURE:
1) Indicate the patient to take a labeled specimen cup, void into the cup and return to
the lab.
2) Wear gloves. Open the container and pour the urine sample into a test tube.
3) Now take a test strip from its container and dip the test strip into the urine. Make
sure all the test squares are immersed in the urine. Take it out and place it on the
table making sure it doesn’t contaminate anything.
4) After 20-30 seconds compare the color of the strip with the color on the strip
container and draw conclusions.

OBSERVATION:
After comparison the following observations were made:

1) Glucose and ketone levels were normal

2) Bilirubin showed an elevation (check for liver function)
3) Blood is present +++
4) The pH is 6
5) Leukocyte level is 70+ and protein level is 15 (0.15)
6) Nitrate is present

Physical examination showed turbid urine, with pungent smell and pale colour.

MICROSCOPIC PROCEDURE:
PROCEDURE:
1) Indicate the patient to take a labeled specimen cup, void into the cup and return to
the lab.
2) Wear gloves. Open the container and pour the urine sample into a test tube.
3) Centrifuge your sample at a 3000 rpm for 5 minutes (make sure to balance
centrifuge to avoid breaking of the test tube).
4) Discard the supernatant and tap tube with index finger to mix sediment with
remaining fluid.
 5) Make a wet mount of sample by transferring 1 drop of material to a slide and
covering with a coverslip.
6) Examine the sample under the microscope under low and high power and draw
conclusions.

OBSERVATION:
 Moving cocci shaped bacteria, pus cell, squamous epithelial cell and red blood cells.
Session 9:

Antimicrobial susceptibility test:

Once a physician has diagnosed the cause of a disease, the next step is to treat it. Treatment of
diseases caused by microbes includes antimicrobial drugs. As many antimicrobials have been
used to treat different species of bacteria some bacteria have development mechanisms of
escaping these treatments. Hence, the necessity of performing antimicrobial susceptibility tests
as a routine procedure in all microbiology laboratories. In laboratories antibiotic disks are used
on culture media containing the specific organism to be tested for its susceptibility.
Antimicrobial susceptibility tests (ASTs) basically measures the ability of an antibiotic or other
antimicrobial agent to inhibit the in vitro microbial growth.

The agar used for this test is the Muller Hinton Agar. This agar is good for the non-
fastidious bacteria. Fastidious organisms which require specific growth supplements need
different media to grow for studying the susceptibility patterns. The selected antibiotic disks
are placed on the surface of an agar plate which has already been inoculated with test bacteria.
During the incubation period, the antibiotics diffuse outward from the disks into the agar. If
there’s no growth of the organism around the antibiotic, it indicates the organism is susceptible
to that antibiotic and the presence of growth around the antibiotic disk indicates the organism
is resistant to that particular antibiotic. The area of no growth is called zone of inhibition. The
diameter of zone of inhibition is measured using a caliper and compared with the standard
chart to obtain the susceptibility and resistance of that particular microbe.

Materials:
1) Inoculation loop
2) Spirit lamp
3) Saline solution
4) Antibiotic disks (Gentamycin (G), Ciprofloxacin (CIP), Chloramphenicol (C))
5) Caliper
6) Incubator
7) Cotton swab
8) Culture of the bacteria to be tested
9) Test tube
10) Muller Hinton Agar
Procedure:
1) Take the cultured bacteria and choose a pure colony. Bring a test tube and add saline
solution to it and place it on the test tube rack. Take a colony using the inoculation loop
(aseptic technique maintained) and emulsify it in the test tube. Mix thoroughly to
ensure the proper mixing and no solid material remains. Keep doing the emulsifying
process until the required turbidity is reached. Check the turbidity using a standard
turbidity card.
2) Take a sterile swab and insert it into the test tube. Gently squeeze the swab on the
inside of the test tube to remove the excess fluid on the swab.
3) Now bring a sterile Muller Hinton agar and streak it according to the usual methods.
4) Let it dry for 5 minutes. ( be careful of contamination)
5) Now using sterile forceps place the antimicrobial plates on the surface of the agar and
gently press them to ensure they are secured. Make sure to keep them apart from each
other so as to not have them interfering with each other’s effect.
6) Cover the plate, invert and incubate it for 24 hours at 370C.
7) After incubation use a caliper to measure the diameter of zone of inhibition. Compare
your measurement with the standard to determine whether the bacteria is sensitive or
resistant to the tested antibiotic.
8) Report your findings.

Observation:
 Both the discs (Ciprofloxacin and Gentamycin) had zone of inhibitions.
Session 10:

Widal and Weil-Felix tests (Slide test):

The Weil-Felix test is used to detect the presence of rickettsia (typhus) infection whilst the
Widal test is used to detect the presence of typhoid (Salmonella typhi and Salmonella
paratyphi). They both detect the level of antibodies in the blood of the patient. The Ag used in
the Weil-Felix test is not from the rickettsia species as they can’t be isolated. It’s rather from
Proteus vulgaris as they both produce antibodies that have similarity. The somatic O Ag is used
in this case. Rickettsia prowazaki and Rickettsia typhi react to OX 19 whilst tick borne typhus
reacts to OX 2 and Oriental species react to OX K. although the best test (gold standard) used in
the typhus scenario is the immunofluorescence test but in resource limited setting this test is
used (has low sensitivity and specificity). Widal test detects the presence of serum
agglutinins/antigens (H and O) in patients’ serum with typhoid and paratyphoid fever. Only the
typhoid Ag are employed here the paratyphoid aren’t as they cross react with the typhoid ones.
O Ag is the somatic antigen whilst H is the flagellar antigen. If the patient has the disease there
will be agglutinations whilst if there is no agglutination the patient is free of the disease.

Materials:
1) Gloves
2) Syringe
3) Citrate coated test tube
4) Centrifuge
5) Test tube rack
6) Test slide
7) Antigen bottles (Ag H, Ag O and Ag OX 19)
8) Micro pipette

Procedure:
1) Wear gloves and draw blood from the patient using a syringe following the aseptic
technique.
2) Drain the blood into citrated bottle/container. Make sure to mix the blood with the
citrate coated bottle to avoid coagulation.
 3) Centrifuge it for 5 minutes at 3000 RPM. Be sure to balance the tube in the centrifuge to
avoid the breaking of the tube.
4) Now carefully take out the tube and place it on the test tube rack. Be sure to avoid the
mixing of the plasma with the rest of the components by not moving it too much.
5) Bring a test card and place it on the table. Label on the top of each circle Ag H, Ag O and
Ag OX 19. Place the respective Ag bottles on the top of the card distant from their
labels.
6) Bring a micro pipette and place two drops of the plasma on the test card. Place three
drops on the three circles individually. Now, add a drop of antigen H on the first circle, O
on the second and OX 19 on the third.
7) Take a tooth pick, individual side for all the three circles and mix them thoroughly until
the whole circle is covered.
8) Rock the slide, gently back and forth and observe for agglutination macroscopically
within one minute.

Result:
 Agglutination on the OX 19 was seen but Ag H and O were clear. Subject
suspected to have Typhus. Further investigation by immunofluorescence
test.
Session 11:

Serology tests for HIV and HBV:

Hepatitis B test is used to detect the presence of the virus in the blood stream using the Ag-Ab
technique as the virus itself is very tiny to be seen via a microscope as is the HIV virus. The
hepatitis B virus has three antigens named: the surface antigen HBsAg, the core Ag HBcAg and
the envelope Ag HBeAg. The core antigen present when there is active viral replication and is
found in hepatocyte biopsy but not in blood. The HBsAg indicates the current infection of the
person by hepatitis B virus whilst the rest two indicate previous infection or vaccine.
Human immunodeficiency virus causes AIDS and has two serotypes 1 and 2. The HIV 1 is
common worldwide whilst the HIV 2 is common in West Africa. Different countries use different
type of kits to confirm HIV results. In Ethiopia the kits used previously were KHB, STAT and
UNIGOLD. Now these have been replaced by the kits known as First Response, UNIGOLD and
VAKA. The necessity of several kits is due to the fact that false negatives and positives tend to
occur in the process of the test. The false positive test obtained by kit 1 should be checked
through by kit 2 and if kit number 2 shows positive, the patient is positive. But is kit number 2
shows negative, we rely on kit number 3 as it will be the deal breaker. The false negative would
be due to recent infection and underdevelopment of antibodies. So the patient should be asked
to come back after three months to be checked again. If he/she test negative he/she is free.
There are two markers on the device the first being the control and the second being the test.
If the device has two lines on both the markers then the test is positive. If it has only one line on
the control the test is negative. But if there’s only one line on the test marker the test is invalid.

Materials:
1) KHB test kit
2) Plasma of the subject to be tested
3) Gloves
4) HBV test kit
5) Micropipette

Procedure:
A) Hepatitis B test kit:
1) Wear gloves and draw blood from the patient using a syringe following the
aseptic technique.
2) Drain the blood into citrated bottle/container. Make sure to mix the blood with
the citrate coated bottle to avoid coagulation.
 3) Centrifuge it for 5 minutes at 3000 RPM. Be sure to balance the tube in the
centrifuge to avoid the breaking of the tube.
4) Now carefully take out the tube and place it on the test tube rack. Be sure to
avoid the mixing of the plasma with the rest of the components by not moving it
too much.
5) Open the HBV test kit and place the device and the dropper on the table.
6) Now using the dropper add 3 drops of plasma at the dropping port of the device.
7) Leave it for 10-15 minutes and record your findings.

B) HIV test kit

1) Wear gloves and obtain plasma of the patient as discussed above.
2) Open the test kit and place the device on the table.
3) Bring a micropipette and introduce 40 microliter at the dropping port of the
device.
4) Apply a drop of diluent to dilute the plasma and enable it to diffuse thru the
sheet.
5) Leave it for 30 minutes.
6) Observe the lines and note your findings.

Result:
 Both the kits showed negative (only one line) on them. Hence, the patient tested
negative for both the tests. For HIV test ask them to come after 3 months