Culture Plating

During the 2nd and 3rd week in the microbiology, I learnt about culture plating, where specimens are inoculated onto agar plates to look for growth of microorganisms. The specimens that are usually cultured in the lab are ;

1) urine culture
2) stool culture
3) throat swabs
4) ear swabs
5) eye swabs
6) sputum culture
7) high vaginal swabs
8) semen culture
9) external/internal wound swabs

For urine cultures, three types of agar plates (Blood Agar, MacConkey Agar and CLED Agar) are needed. Splitting each of the plates into four quadrants, a 1-microlitre loop is used to dip into the urine and then streaked onto the plates. The Blood Agar is made up of 5% Sheep Blood and is used for the isolation and cultivation of nonfastidious and fastidious microorganisms. Most importantly, it is used to determine if the microorganism is of pure of mixed culture. MacConkey Agar is a differential plating medium recommended for use in the isolation and differentiation of lactose-fermenting organisms from nonfermenting Gram-negative enteric bacteria. It is selective by the presence of specific inhibitors. A Cystine Lactose Electrolyte Deficient (CLED) Agar is a valuable non-inhibitory growth medium used in the isolation and differentiation of urinary organisms. Being electrolyte deficient, it prevents the swarming of Proteus species. Lactose fermenters produce yellow colonies on CLED agar while non-lactose fermenters appear blue.

Stool cultures are inoculated on a xylose lysine deoxycholate (XLD) plate, to check for presence of Salmonella enterica. It is formulated to inhibit Gram-positive bacteria, while the growth of Gram-negative bacilli is encouraged. The colonies of lactose fermenters would appear yellow.

For specimens obtained from the respiratory tracts, such as throat, ear, eye and sputum cultures, they require the same media. A Blood Agar, MacConkey Agar and a cooked meat broth is needed. A cooked meat broth is used to revive the microorganisms present in the specimen.

Semen cultures are inoculated on a Blood Agar, MacConkey Agar and a Chocolate Agar. A chocolate agar is used to isolate for any presence of Gonococci, which causes Neisseria gonorrhoeae, a sexually transmitted disease.

High Vaginal swabs requires to be inoculated on a Blood Agar, MacConkey Agar and a Sabouraud Agar. A Sabourad Agar is a medium used to cultivate pathogenic and nonpathogenic fungi. In this instance, a formation of creamy colonies on the plate is an indication of candida, which is a sign of fungal infection.

For wound swabs, we would have to determine whether it is obtained internally from the body or externally. For external wound swab, a Blood Agar, MacConkey Agar and cooked meat broth is needed. For internal wound swab, an additional Blood Agar meant for ANO2 incubation is needed. This is to see if the organisms is able to thrive without oxygen.

Haem- Bone Marrow

The technologist’s task in bone marrow aspiration procedures is to carry out on-site smearing of patient’s marrow. Usually there are 3 different types of smears done:

1) Direct aspirate/ wedge smears:
-Performed by placing a few marrow particles at one end of a clean slide and smearing with a spreader.
-Marrow particles (fragments) must be found mostly at the tail of the smear.
-After staining slides with MayGrunwald Giemsa stain, the fragments appear bluish in colour.
-When viewing under a microscope, a trail of cells can be seen following the fragments. Differential counts are performed in these cellular trails.
-Fragments are examined to determine the cellularity.

Wedge Smear

2) Squashed Preparation
-A few particles (fragments) are firstly placed on a slide. Another slide is then gently compressed on the first slide to spread and disperse the particles.
-The crushed fragments will be at the center of the smear.
-To see the cells of the fragments more clearly. (since the fragments are more spread out when it is squashed)

3) Trephine Imprint (from bone)
-Several (2-4 slides) touch or imprint preparations were made by gently touching the core plug (bone) and rolling it between 2 slides.
-This smear or preparation is especially useful if the aspirated marrow is diluted or a “dry tap” (i.e. no marrow particles or fragments can be extracted) is obtained.
-When ‘rubbing’ the core plug between slides, the cells on the surface of the core plug will be attached to the slide.

Other possible presence of cells to take note or when viewing marrow slides:
-Nucleated red blood cells (nRBCs)
-Megakaryocytes
-Mitotic cells (undergoing division)

Confirmatory tests in relation to possible diagnosis:
1) Iron stain (demonstrate presence of hemosiderin/storage iron in erythroblasts/sideroblasts or erythrocytes): useful in the classification of anemia associated with defective Hb synthesis. Can also be used to identify presence of ringed sideroblasts in the diagnosis of soderoblastic anemia and myelodysplasia.

2)Neutrophil Alkaline Phosphatase (NAP score): Used nto distinguish chronic granulocytic leukemia (CGL) from other myeloproliferative disorders and Polycythaemia Rubra Vera from secondary polycythaemia.

3)CSF test for blast cells: used to examine leukaemic cells in the CSF from a cytospin preparatrion. Essential in the diagnosis of meningeal leukaemia. Also used to monitor the adequacy and effectiveness of treatment.

4) Periodic Acid Schiff (PAS) reaction : stains a variety of carbohydrates including glycogen (found in haemopoietic cells). Used in differential diagnosis of acute leukaemia (e.g: Lymphoblasts are PAS positive.

UFEME

I am currently attached to the microbiology section and it has to be said that there are alot more tests that are conducted here compared to what we learnt school. While we mostly carry out inoculation and streaking of microorganisms that has been commercially prepared in school, we are able to handle real specimens such as urine, stool, semen and vaginal swabs in a real-time working environment. For this post, I will be writing about urine analysis. A simple routine test to detect any abnormalities in the urine is the Urine Full Examination Microscopy Elements (UFEME). It involves the urine to be pipetted into a Kova glass slide chamber, where it will then be viewed under a microscope for the presence of any abnormalities. Also, it requires the dipping of a urine dipstick into the urine bottle and then placing it onto a urine analyzer, where it will process the result of the urine components.

The components consists of:

1) pH - measures the urine acidity or alkalinity
2) Specific Gravity - measures the amount of substances dissolved in urine
3) WBC count - presence would indicate urinary tract infection, cancer or kidney infection
4) RBC count - presence would indicate inflammation/infection to bladder, kidneys or ureters
5) Ketone - large amounts may be an indication of diabetic ketoacidosis
6) Glucose - large amounts may be a sign of diabetes or damaged/diseased kidneys
7) Protein - presence may be caused by kidney damage, fever or normal pregnancy
8) Nitrite - presence would indicate urinary tract infection

During microscopy analysis of urine, the following materials are usually looked out for.

1) Red blood cells
2) White blood cells
3) Casts
4) Crystals
5) Yeast cells, bacteria or parasites

A healthy person should not have any presence of the materials stated above although a little amount or RBCs, WBCs or crystals can still be considered normal. However, a person that has a large amount of presence of any of these materials would most likely be infected with a urinary tract infection (UTI). Red blood cells in the urine may be caused by kidney or bladder injury, kidney stones, UTI, inflammation of the kidneys (glomerulonephritis), a kidney or bladder tumor, or systemic lupus erythematosus (SLE). White blood cells (pus) in the urine indicate UTI, bladder tumor, inflammation of the kidneys, systemic lupus erythematosus (SLE), or inflammation under the foreskin of the penis or in the vagina.

Depending on the type casts, they can indicate inflammation or damage to the tiny tubes in the kidneys, poor blood supply to the kidneys, metal poisoning (such as lead or mercury), heart failure, or a bacterial infection.

Excessive amounts of crystals, or the presence of certain types of crystals, can indicate kidney stones, damaged kidneys, or problems with metabolism. Some medications and certain types of urinary tract infections can also increase the number of crystals in urine.

Bacteria in the urine indicates UTI. Yeast cells or parasite, such as trichomonas vaginalis can indicate an infection of the urinary tract.

Haem case study 2

Case: Thalassemia-related disease

Sample Collection: Whole blood via venipuncture
Samples Processing: Blood sample is collected in an EDTA tube (there must be at least 3mL of blood). Once received, a lab no. is given to the sample for identification purposes.

Preliminary laboratory tests: Alkaline Cellulose Acetate Electrophoresis (ACAE), Variant test.
The ACAE test is used to screen for Hemoglobin (Hb) variants. Hb have a net negative charge at pH 8.6 and move towards the positive electrode in an electrical field. The variation in the amino acid content of the different Hb causes variation of net charge of each Hb type. This hence determines their rate of mobility. ACAE makes use of cellulose acetate strips. When run at 350 volts for 25 minutes, the Hb bands will appear accordingly on the strips.
Procedure:
1) Blood samples are firstly washed with 0.9% NaCl and centrifuged. This step is done 2 times. The purpose is to get packed cells (pellet).
2) A fixed amount of washed packed cells is then pipetted into test tube containing hemolysate reagent.
3) The mixture is mixed and left to stand for 20 mins.
4) Meanwhile, prepare the cellulose acetate strips. It is labeled accordingly and placed in a carrying rack. The rack is then slowly lowered (to prevent formation of air bubbles) into a container containing supre-heme buffer and soaked for 5 mins.
5) Prepare sample wells. The patients’ hemolysate mixture (from step 3) and also the control (ESFA) is placed into each sample wells.
6) The applicator is primed by pressing gently the tips into sample wells 1-2 times. Check if the samples are well picked up by the applicator by applying it on filter paper.
7) It is then applied to the cellulose acetate strip.
8) The strip is then quickly placed in electro chamber (containing supre-heme buffer).
9) Electrophoresis the plate for 25 mins at 350 volts.
10) Staining: Ponceau S stain for 5 mins, 5% acetic acid for 2 mins, absolute methanol for 2 mins, cleaning solution for 5-10 mins.

Interpretation:
If there’s high amount (dark band) of Hb A2, this may indicate beta thalassemia as since there is lack of beta-chains, there’ll be excessive alpha chains causing high amounts of Hb A2. Confirmatory tests should follow.

An example of a gel strip.

The variant test is used to quantitate and detect HbA2 and HbF. It is intended for the separation and area percent determination of HbA2 and HbF, and also assists in the identification of abnormal Hb in whole blood using ion-exchange high performance liquid chromatography (HPLC).

Preliminary investigation: Haemoglobin levels, HbA2, HbF

Confirmatory tests:
1) Sickling test (can be used if the HbS band appears in ACAE test)
- Gives a rapid screening test for the Hb S status of a patient.
- Demonstrate the sickling phenomenon in test samples. Sickling phenomenon occurs with low oxygen tension. A small drop of blood is added to a reducing agent. It is then sealed between slide and cover glass, and incubated at 37 degree Celsius for sickling to occur. A negative and positive control is included together with the other samples. If there is no sickling in the positive control, this indicates false negative result hence test have to be repeated.


Arrows indicates sickle cells.

2) Acid elution test (Kleihauer)
- Used to determine the distribution of fetal Hb in red cells (confirmatory test when there’s an unusual Hb F band in the ACAE test.)
- Hb F resist acid elution while Hb A is denatured under these conditions. Thin blood films are exposed to low pH acid buffer, washed and then stained. Cells containing Hb F are deeply stained while cells containing Hb A gibe a ghost-like pale appearance.

3) Acid gel electrophoresis
- Enables separation and provides confirmation of Hb variants that co-migrated in alkaline Hb-electrophoresis. (e,g: to differentiate Hb C from Hb E when theres a dark band indicating Hb E in ACAE test).
- Hemolysate is applied to an acid buffered agarose gel. Electrophoresis then takes place. Following that, the Hb in the gel is immobilised in a fixative solution and the gel is dried to a film. To see the Hb pattern clearly, the gel is stained with a protein specific stain.

4) Hb H test
-The Hb-H test is performed to screen for Hb-H inclusion bodies. It can be used as a confirmatory test in the diagnosis of alpha-thalassemia.
-2 drops of brilliant cresyl blue stain is added to 100 microlitres of blood. After incubation period of 1 hr at 37 degree celcius, samples are left to cool before smears are made for microscopy viewing. Presence of Hb-H is indicated by “golf-like” cells.

FBC parameters

During my third week at Haematology, I was assigned to the "processing" section, where I am responsible for the handling of EDTA blood tubes. After the medical technologists have verified the blood samples, these tubes would be run on the Full Blood Count analyzer. The machine at my workplace is called the Beckman Coulter LH 750 Hematology Analyzer (picture below). It performs automated cell counting and immediately displays results for the blood sample's FBC on the LIS.

There is also a manual hematology analyzer called Beckman Coulter Act Diff (picture below), which is used only to re-run samples that have abnormal readings.

Below are some of the FBC parameters which I have to take note of should any of the value of the sample results fall within any of the range.


1) Hb <9.0>18.0 g/dL

Action to be taken :
- Re-run sample at Coulter Act Diff
- Make a peripheral blood smear
- Print report

2) Platelet <140>600 x 10^9/L

Action to be taken :
- Re-run sample at Coulter Act Diff
- Make a peripheral blood smear
- Print report

3) WBC <3.0>30 x 10^9/L

Action to be taken :
- Re-run sample at Coulter Act Diff
- Make a peripheral blood smear
- Print report

4) Hb <7.0>18.0 %

Action to be taken :
- Re-run sample at Coulter Act Diff
- Print report

5) RBC >5.0 x 10^12/L + MCV <75 fL + MCH < 25 pg

Action to be taken :

- Make a peripheral blood smear
- Print report

6) Eosinophil Count > 6.0 %

Action to be taken :
- Print report

7) Platelet Clumps Remarks

Action to be taken :
- Make a peripheral blood smear
- Print report

8) Verify differential count flagging

Action to be taken :
- Re-run sample at Coulter Act Diff
- Print report

9) NRBC > 8.0%

Action to be taken :
- Make a peripheral blood smear
- Print report


The principles behind re-running a sample in another analyzer is to compare both the results from the automated and manual analyzer. This is to confirm if the values for the same parameters tally, even if they do differ slightly. Also, making a peripheral blood smear of the sample allows the viewing of the morphology of the cells under the microscope. For example, it is to check if the blood is really microcytic or hypochromic. Upon validating the results of the blood tubes, they are then sorted out according to the test required to be performed, such as Blood Group, Erytrocytes Sedimentation Rate or Malaria Parasite.

Haem case study

Female, 57 years old
Given diagnosis: Acute lymphocytic leukaemia (ALL)

Routine FBC / Reference range
WBC --> 2.16 X 103 cells/mL /4.5 – 11.0 X 10(power 9)
RBC --> 3.04 X 106 cells/mL /4.2 – 5.4 X 10(power 12)
Hb --> 9.5 g/dL /11.5 – 16.0
HCT --> 27.2% /37% – 47%
MCV --> 89.7 fL /80 -96
MCH --> 31.3 pg /27-29
MCHC -->34.9 g/dL /31.5 – 34.5
RDW --> 15.6% /11.5% - 14.5%
PLT --> 21 X 103 cells/mL /150 – 450 X 10(power 9)

Routine WBC differential (100%) /Reference range
Neutrophil : 38 /40-75
Lymphocyte: 47 /15-41
Monocyte: 15 /2-10
Eosinophil : 0 /0-6

Morphology flags
Blasts : +
ATYP(atypical lymphocyte): +

Discussion:

Clinical presentation/ symptoms : Fatigue, weight loss, anemia, bleeding gums, breathlessness, enlarged lymph nodes, liver and/or spleen, fever and paleness.
Sample Collection: Whole blood via venipuncture
Samples Processing:
1)Blood sample is collected in an EDTA tube
2)A blood smear is made followed by staining. This is for microscopic viewing.
3)The blood sample is then placed on a special rack on the automated analyser to carry out full blood count (FBC)

Preliminary laboratory tests: FBC
Preliminary investigation: RBC, WBC, Hb, HCT, MCV, MCH, MCHC, RDW, PLT, differential count and morphology flags.
Expected results for laboratory tests:
1)Values of WBC, RBC, Hb, HCT and PLT are lower than reference range
2)Values of MCH and RDW are higher than reference range
3)Values of MCV and MCHC are normal.
4)There are also abnormal amounts of neutrophils, lymphocyte and monocyte.
5)There are presence of blasts (lymphoblasts) and atypical lymphocyte (ATYP)

Interpretation:
1) Values of WBC and PLT are low, indicating that there is failure of the bone marrow. In ALL, the WBC is usually high. In this case, the patient may have gotten leukaemia for quite some time hence the WBC may decrease due to earlier treatment.
2) The low amounts of RBC, Hb, HCT indicates anemia, which is one of the symptoms of leukaemia.
3) The presence of blasts highly indicates possibility of ALL.
4) An increased level of RDW reveals presence of anisocytosis due to bone marrow failure.
5) The abnormal amounts of WBC differential count confirm that there is abnormality of the WBC.

Confirmatory tests:
1) Cell markers test (flowcytometer) to confirm presence of lymphoid or myeloid.
2) Cytochemical stain: Used in the identification of the different subtypes of acute leukaemia.
3) Immunological markers (Antigens/ antibodies specific to ALL): A positive test indicates presence of ALL
4) Bone marrow aspiration / biopsy: An increased number of cells and lymphoblasts confirms presence of ALL.

Prognosis: There are treatments for leukaemia. This includes chemotherapy, steroids, radiation therapy and growth factors. The main goal of treatment is remission of the cancer. Administration of blood products (packed RBCs, platelets) may also be needed to treat the anemia and low platelet count.