Home Work for Intern
1. Standard operating procedures for blood culture
a. How are samples processed
-Examine the blood culture bottles to see if the information on the bottle correlates with
the information on the requisition form.
-Check blood culture sample against rejection criteria.
-Label the blood culture bottle with the next available internal laboratory number.
-On the top right-hand corner of the requisition form, the type of sample that is sent is
written before the internal laboratory number eg. BH76
-Label a triplate (BA, Mac, and CB) and a clean slide with the assigned number.
Venting of Blood culture Bottles
-Decontaminate the septum of the bottle with 70% alcohol starting from back to front.
-Insert the venting needle through the septum.
-Mix the blood culture, remove the cap from the needle using a forcep.
-Invert bottle over container with 10% bleach to get free flowing drops of blood,
inoculate a drop of blood to the triplate and labelled slide for gram stain.
-Streak plate for Isolation.
-Discard needle using forcep and incubate plate in co2 condition and inspect daily for
7 days. Macroscopic examination includes visually inspecting the bottle for signs of
turbidity, gas formation and hemolysis. Place slide on slide warmer for staining.
b. Rejection criteria specific to blood culture
-Blood Culture specimens:
-. Kept for over 48 hours at room temperature
-. That have been refrigerated
- Clotted samples for culture and sensitivity (except clot culture accepted for S.
Typhi)
-. Expired blood culture bottles
- If the samples coming from KPH or VJH ensure that they have a ward number on the
form if not thats a rejection sample
c. Any other special consideration given when processing blood culture samples
-. Big bottle for the manual method are not accepted only the small baby bottle will be
process.
- All blood culture samples that meet the 24 hours criteria are processed on the same day.
If the 24-hour criteria is not met, a cap is placed over the bottle and incubated.
- At the end of 7 days the bottles are subculture regardless of visual reading.
2. Standard operating procedures for CSF
a. How are samples processed
-Examine the CSF sample to see if the information on the sample correlates with the
information on the requisition form. Example the JPL number
-Check CSF sample against rejection criteria.
-Describe the appearance of CSF sample eg. Bloody, clear, xanthrochromic, turbid.
-Place the barcode on the Triplate (BA, Mac, and CB) and label a clean slide soaked in
70% alcohol with the four last digit of the JPL number.
-Using a clean pipette, place a drop of CSF on the Triplate and on the slide for gram stain.
-Streak for isolation. Incubate the plates in carbon dioxide for 24 hours. (Procedure
conducted under fume hood).
b. Rejection criteria specific to CSF culture
- Sample that have been sent or put into an refrigerated is rejected as it is not the correct
temperature for incubation
- Kept for over 24 hours at room temperature
-Any other special consideration given when processing CSFculture samples
-
3. Standard operating procedures for Stool Culture
a. How are samples processed
[Link] the blood culture bottles to see if the information on the bottle correlates with
the information on the requisition form.
[Link] blood culture sample against rejection criteria.
b. Rejection criteria specific for stool culture
[Link] formalin for culture and sensitivity
b. In a sterile dry container, that are more than 2 hours old at room temperature or that are
more than 24 hours old at 2-8.C
c. Dry rectal swabs and rectal swabs in transport medium more than 24 hours at room
temperature
d. In transport media, that are more than 48 hours at room temperature and those in e.
transport media (plycerol saline) that have changed from pink to yellow in colour.
Stool contaminated with water, soil or urine
[Link] containers with disinfectants, detergents or any additives to prevent
c. Any other special considerations
-Samples that are greater than 1ml in volume are centrifuged.
-Bloody samples are not centrifuged.
-India ink is performed on patients >12 years or on request.
4. 10 Quality control measures taken in the department for the processing of each sample type
a. Processing of Stool Samples
-Examine the stool sample to see if the information on the sample correlates with the
information on the requisition form.
-Check stool sample against rejection criteria.
-Describe the appearance of stool sample eg. Bloody, watery, formed, semi-formed.
-Place the barcode on the Triplate (XLD, Mac) and label the Gn broth with the four last
digit of the JPL number.
-Use a cotton tip swab to make a pool and to inoculate the Gn broth. Streak for isolation.
- Plate is placed in O2 conditions for 24 hours. (Procedure conducted under fume hood).
b. Rejection Criteria
- In a sterile dry container that is more than 2 hours old at room temperature.
- Dry rectal swabs and rectal swabs in transport medium more than 24 hours at room
temperature.
-Stool contaminated with urine, soil or water
-Stool contaminated with disinfectants, detergents
- In formalin for culture and sensitivity.
4. Quality control measures taken when processing the sample.
- Always check the plate with the blood sample before streaking the plate.
- The gloves are changed before decontaminating the septum of the blood culture bottle to
prevent contamination.
- The CSF slides are flamed to maintain sterility.
Major Pathogens based on sample type
CSF
1. C. Neoformans
What is the appearance of a SF sample with suspected C. Neoformans?
a. Agar used in identification
-The organism will appear as brown to black pigmented smooth colonies.
b. Colony morphology of C. neoformans on Media
- The organism can appear as single, budding, thin- walled, round cells containing Gram
positive granular inclusions.
c. Gram stain appearance C. neoformans
- gram positive budding yeast cell
d. Flow chart work up of identification
Inoculation of primary culture media observe for mucoid colonies perform India
ink positive rapid urease test + germ tube Urease test +ve and germ tube -ve
inoculate bird seed agar (isolates will appear as brown to black pigmented smooth colonies)
Confirmatory test cryptococcal antigen testing
e. Pathogenesis include
i. How is the organism introduced to host cells
-
ii. Source of infection
- Source of infection- Immediate contact with pigeon droppings or unwashed raw fruit.
iii. Definition of vomocytosis
-ii. Vomocytosis- the expulsion of pathogens or organisms that have previously been
engulfed by a phagocyte, leaving both cells undamaged.
iii. Definition of intracellular opportunistic pathogens
a. Virulence factors
- Polysaccharide capsule – The capsule protects the bacteria from engulfment by
macrophages thus enhancing the ability of the bacteria to cause disease.
- Phenol oxidase- It is use to synthesize melanin which is used as an antioxidant which
enhances the virulent ability of the organism.
i. incubation period
- The incubation period for [Link] is usually 50 days.
ii. how does the pathogen manifest itself in the central nervous system
-
ili. Main symptoms of an infection in the central nervous system
-Main symptoms in the central nervous system include headache, fever and neck ache.
iv. Treatment
-The administration of antibiotics such as amphotericin B in combination with flucytosine.
v. Prevention
-It can be prevented by so avoiding areas that contain bird feces , dust that contains any type
of bird feces may also help prevent infections.
Blood culture
1. Common GPC isolated from Blood Culture
- Common Organisms isolated-S. aureus
2. Common GNB isolated from Blood Culture
- Common Organism [Link], Klebsiella Pneumoniae
Stool culture
3. S. Aureus
i. Appearance of stool sample with suspected S. Aureus
- Stool samples suspected with S. aureus as a watery appearance.
ii. Agar used for identification
- The agar that can be used to isolate and identify S. Aureus is Mannitol salt agar.
1. Colony morphology on media
- the organism will appear as yellow colonies.
2. Gram stain appearance
- The isolate will appear as a gram-positive cocci upon gram stain.
3. Flow chart workup of identification
catalase test Coagulase (+,
Inoculation of Gram stain
clumping /
MSA (gram positive (+)
agglutination
cocci)
Positive seen).
[Link]
Pathogenesis
a. Source of infection
- i. Source of infection- The consumption of foods that are not properly cooked.
b. Define enteritis
- Enteritis- The inflammation of the intestine particularly the small intestine caused
by consuming foods contaminated with pathogenic microbes.
c. Virulence factors
- S. aureus produces cytolytic toxins that lyses red blood cells (hemolysins) and
white blood cells (lekotoxins) in the host. Staphylococcal enterotoxin is also
produced by the organism. It is responsible for stimulating cytokine release and
inflammation.
i. Type of toxins used by the organism
a. How does the pathogen manifest itself in the Lower GI Tract
-
b. Incubation period
- The incubation period for salmonella is usually 2-4 hours.
c. Main symptoms of an infection in the digestive system
- Main symptoms in the digestive system include cramps in stomach, vomiting,
nausea, diarrhea, fever or chills
d. Treatment
- Rest and the administering of IV fluids to help prevent dehydration and fatigue.
e. Prevention
- It can be prevented by- washing hands frequently, thoroughly cooking meat and
avoiding unpasteurized milk.
Salmonella
a) Appearance of stool sample with suspected Salmonella infection
Stool appears watery and it may have blood or mucus
b) Agars used for identification
XLD and MacConkey are used for investigation
c) Colony morphology on media
On XLD agar colonies appear red with black center indicating H2S production and on
MacConkey colonies appear colorless indicating non lactose fermenting
d) Flow chart workup of identification
Inoculation XLD agar, Observe for red colonies with black
MacConkey agar and centres, clear non lactose fermenting
GN broth colonies and Growth in GN broth
Use colonies on XLD agar using Kligler shows K/A, H2S and gas variable Urease
Kligler iron agar and urea negative tube remains yellow. Further biochemicals
biochemical tube. Use GN broth to are done citrate, MIL and PPA tube. Perform
inoculate XLD and MacConkey agar carbohydrate fermentation using Malonate,
maltose, D-Mannitol, lactose and sucrose using
isolate from XLD agar
Citrate positive blue colour, motility positive,
indole negative, lysine positive and PPA
negative. Malonate -ve, maltose +ve, D-
mannitol +ve, lactose -ve and sucrose -ve.
Serotype using O, H and VI antigen
O +ve, H +ve ad VI -ve – typical Salmonella O
+ve, H +ve and VI +ve – Salmonella Typhi
e) Types of antigens present and characteristics of them
O antigen (somatic antigen) – a part of outer membrane of bacterial cell wall comprised of
specific sugar molecules. This allows for the identification of different salmonella serotypes.
H antigen (flagellar antigen) - allows for motility of organism thus enhancing its contact and
dissemination in host.
VI antigen (capsular antigen) – polysaccharide capsules are protective surface layers that
enhance virulence.
f) How many serovars of salmonella are there
There are more than 2500 serovars of salmonella.
g) Pathogenesis
Salmonella infection is usually caused by the consumption of undercooked meat
(beef), dairy products (egg, milk) as well as foods that are contaminated with feces
from an infected animal
h) Virulence factors
i) Incubation period
j) Source of infection
k) Treatment
l) Prevention
5. Shigella
a) Appearance of stool sample with suspected
b) Agars used for identification
c) Colony morphology on media
d)Flow chart workup of identification
e) Types of antigens present and characteristics of them
f) How many serotypes of shigella are there?
g) Pathogenesis
h) Virulence factors
i) Incubation period
j)Source of infection
k)Treatment
l) Prevention
6. E. Coli 0157:H7
1. Appearance of stool sample with suspected
2. Agars used for identification
3. Colony morphology on media
Flow chart workup of identification
Types of antigens present and characteristics of them
Pathogenesis
Virulence factors
Incubation Period
1. Source of infection
2. Treatment
3. Prevention
Leanna Lewis
