CASE 1: Mr. Watson- Her, PERITONEAL PUS

Case History: Five days ago Dr. Noital performed an emergency appendectomy patient. Post-operative fever, neutrophilia, and severe pain were noted and a subsequences. exploratory laparotomy has revealed a small nick in the small intestine together with, extensive peritoneal pus. The nick has been repaired and the peritoneum flushed With saline. A swab of the pus has been transported to the laboratory. A smear of the pus has already been Gram stained and examined for you, please see results in table below.

MICROSCOPIC EXAMINATION (Gram stain):

+++ to indicate more than 8 human cells per oil immersion field (x100),

++ to indicate 5-8 and + to indicate fewer than 4.

** +++ to indicate more than 20 bacteria per oil immersion field (x100),

++ to indicate 10-20 and + to indicate fewer than 10.

PRACTICAL ONE:

a) Use the swab provided to make a primary inoculum on the following media, in the order given below and use a streak technique, using your loop (see techniques section), for single colonies.

Clearly label each plate: Group ID, class ID, case number (i.e. C1), and date. Discard your swab once used.
- HBA, incubate aerobically 24 hrs  37°C*
- MAC, incubate aerobically 24 hrs 37°C*
- (HBA+Neo) and incubate anaerobically for 48 hrs 37°C*

* Note: The laboratory staff will remove these from the incubators and store these at 4°C until your next session.

Practical Two:

Examine all the culture plates for growth. Look carefully to see if there Is more than one type of bacteria. Describe and quantify (+, ++, +++).

If there is growth on the MAC plate, note whether it is a Lactose fermenter (pink). Perform a direct gram strain from the HBA and HBA +Neo plates.

*The "2" and "4" column should only be used if more than one type of bacteria is found. Otherwise leave these columns blank.

a) All organisms suspected of being anaerobes must be subcultured on to 2 HBA plates, and incubate one aerobically and the other anaerobically to confirm that they will not grow in the presence of oxygen. Identify any anaerobic bacteria further (see ID key)

(b) All organisms should then be identified by using the Gram negative or Gram positive Identification Keys provided in the last 2 pages of the manual. Follow the key by carrying out any tests required to move on to the next step until the organism has been identified.

Descriptions of each test and the methods can be found in the "Techniques and Tests" section of this manual.

Record all tests setup, observations and results in the tables provided. If you are not sure whether you have a PURE culture to set up your tests, set up a purity plate (streak plate)

c) Once you have identified the aerobic or facultative anaerobic isolate/s, and if you think the organism/s could be of significance, you need to set up susceptibility test to antibiotics.

What are the implications for your results, if the antimicrobial susceptibility results for the quality control organism do not fall within the expected range (as per Table in techniques section)?

CASE 4: Mrs.Juan Sagan, URINE SAMPLE

(PROVIDED IN PRACTICAL ONE)

Case History: Mrs. Juan Sagan is experiencing frequency of urination, a burning sensation when passing urine, back pain, bloating and abdominal pain. She is 24 years old and has just returned from her honeymoon in the Maldives. Dr. Noital provides you with a midstream specimen of urine.

MICROSCOPY: 5 X 10⁴ white blood cells/ mL
                       2 X 10² RBC/ mL
                       Nil epithelial cells/ ml
                       +++ Bacteria

PRACTICAL ONE:

a) Using the midstream specimen and a calibrated loop, inoculate a HBA plate (see technique section).

b) Dilute the urine specimen 10^-1, 10^-2, 10^-3 and 10^-4 in 9mL saline tubes provided (see technique section). LABEL THE BOTTLES. DO NOT LABEL THE LIDS. Discard urine once used

c) Accurately measure 100 μL of each of the last three dilutions, and spread over the surface of a 3 HBA plates using a sterile disposable spreader, one plate for each dilution.

d) Place one drop of the 10^-3 dilution on a section of a MAC plate and streak out for single colonies

e) Accurately measure 100 μL of the 10^-3 dilution, and spread carefully over the surface of the Chromogenic Urinary Tract Infection (UTI) agar plate, using a sterile disposable spreader.

f) Incubate at 37ºC aerobically for 24 hrs.

Practical TWO:

For serial dilution plates: count the colonies on the HBA plate, (only d there are between 30-300 colonies). Determine the number of bacteria/ml of urine. ( = number of colonies/volume used (in mL dilution factor)

For Calibrated Loop: count the number of colonies on the MBA plates and determine the CFU/mL.

The number of CFUs is multiplied by 1000 to determine the number of microorganisms per millilitre in the original specimen, CFU/mL.
Isolate a single colony and prepare a smear for a Gram stain

Examine and record colony appearance on the MAC and UTI plate.

Identify any Gram-negative or Gram-positive bacteria using the simplified keys provided. Follow the appropriate key by carrying out any tests required to move on to the next step until the organism has been identified. Details of tests will be found in the back section of this manual.

Record all tests set up, observations and results on the tables provided. If you are not sure whether you have a PURE culture to set up your tests, set up a purity plate (streak plate)

What are the implications for your results, if the antimicrobial susceptibility results for the quality control organism do not fall within the expected range (as per Table in techniques section)?