NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL IMMUNOLOGY (HCB 21303)

Blood smear, blood film and reaction between bloods and Candida albicans
Objectives: 1. To learn the techniques to prepare the blood smear and blood film. 2. To learn the technique of how to do the Gram and Giemsa staining. 3. To observe the reaction between white blood cells and Candida albican. 4. To learn the components of the blood. Introduction: A blood film or peripheral blood smear is a microscope slide made from a drop of blood, which allows the cells to be examined microscopically. Blood films are usually done to investigate haematological problems (disorders of the blood) and, occasionally, to look for parasites within the blood such as malaria and filaria. Thick films allow the microscopist to screen a larger volume of blood and are about eleven times more sensitive than the thin film, so picking up low levels of infection is easier on the thick film, but the appearance of the parasite is much more distorted and therefore distinguishing between the different species can be much more difficult. Giemsa stain is a differential stain. It can be used to study the adherence of pathogenic bacteria to human cells. It differentially stains human and bacterial cells purple and pink respectively. It can be used for histopathological diagnosis of malaria and some other spirochete and protozoan blood parasites. Giemsa stain is a classical blood film stain for peripheral blood smears and bone marrow specimens. Erythrocytes stain pink, platelets show a light pale pink, lymphocyte cytoplasm stains sky blue, monocyte cytoplasm stains pale blue, and leukocyte nuclear chromatin stains magenta. Candida albicans is a diploid fungus and a causal agent of opportunistic oral and genital infections in humans. Systemic fungal infections (fungemias) have emerged as important causes of morbidity and mortality in immunocompromised patients.

NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL IMMUNOLOGY (HCB 21303)

Candida albicans is commensal and is among the gut flora, the many organisms which live in the human mouth and gastrointestinal tract. Under normal circumstances, Candida albicans lives in 80% of the human population with no harmful effects, although overgrowth results in candidiasis. Candidiasis is often observed in immunocompromised individuals such as HIV-positive patients. Candidiasis also may occur in the blood and in the genital tract. Candidiasis, also known as "thrush", is a common condition which is usually easily cured in people who are not immunocompromised. To infect host tissue, the usual unicellular yeastlike form of Candida albicans reacts to environmental cues and switches into an invasive, multicellular filamentous forms. This experiment is about the reaction between the white blood cells and Candida albican. Materials: Lancet, alcohol swab, slides, microscope, oil immersion, crystal violet, Grams iodine, 95% ethanol, safranin, Giemsas stain, Candida albican, microtiter plate, micropipette. Methods: A. Blood smear and blood film preparation. 1. The slides were prepared. 2. A finger was swab with alcohol swab then it was prick using a lancet. 3. Then, the blood was dropped on the center of one slide. A corner of the other slide was used to spread the drop in a circular pattern until it is the size of a dime. 4. A proper density of blood film is when the slide was placed over newsprint; the words are barely to read. 5. Then the slide was lay flat to allow the smear to dry thoroughly. 6. After that, another two slides was prepared. A drop of blood from the same puncture site was dropped on the edge of one slide.

NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL IMMUNOLOGY (HCB 21303)

7. The other slide was used at 30-45 angles up to the drop; allow the drop to spread along the contact line of the 2 slides. The upper slide was quickly pushed toward the other end of the lower slide. 8. The smear was assured to have a good feathered edge. Then the blood smear was let dried at room temperature.

B. Staining of the blood smear using Grams stain. 1. After the blood smear was dried, a few drop of crystal violet was dropped on top of the slide and covered the smear. Then it was let stand for 1 minute. 2. Then after 1 minute, wash the slide with tab water, then a few drop of Grams iodine was added and let stand for 1 minute. Then it was washed. 3. Then a few drop of 95% ethanol was dropped and let stand for 30 seconds then it was washed quickly. 4. Then a few drop of safranin was dropped on top of the slide and let stand for 1 minute. Then it was washed and dried. 5. Then the slide was observed under the microscope from low magnification to high magnification. The result obtained was drawn.

C. Staining of the blood film using Giemsas stain. 1. After the blood film was dried, a few drop of Giemsas stain was dropped on top of the slide and let stand for 3 minutes. 2. After 3 minutes it was washed under the tap water. Then it was dried. 3. Then the slide was observed under the microscope from low magnification to high magnification. The result obtained was drawn.

D. Reaction between the blood and Candida albican.

NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL IMMUNOLOGY (HCB 21303)

1. The blood was pipetted then a drop of Candida albican was added into the blood then it was mixed. 2. Then it was incubated for 5 minutes. 3. After 5 minutes, the blood was smear on the slide and was stain using Grams staining. 4. Then after dried it was observed under the microscope from low magnification to high magnification and the results obtained was drawn.

Results:

Blood smear Discussion:

Blood film

Blood smear with Candida albican

From the experiment, the result shows the structure of the blood smear and blood film of normal and healthy blood sample when being observed under microscope. In blood smear, it was clearly showed the featured of red blood cells, white blood cells and platelets. However, in blood film, the blood components cannot being observed clearly because the structure of the red blood cells, white blood cells and platelets were destroy and it was mixed together. The white blood cells (WBCs, leukocytes), red blood cells (RBCs, erythrocytes), and platelets (thrombocytes) are produced and mature in the bone marrow and are

NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL IMMUNOLOGY (HCB 21303)

eventually released into the bloodstream as needed. WBCs main function is to fight infection, while RBCs carry oxygen to the tissues. Platelets appear as small cell fragments and, when activated, form a plug as one of the first steps in blood clotting. The number and type of each cell present in the blood is dynamic but generally maintained by the body within specific ranges. Values can fluctuate at times of illness or stress; intense exercise or smoking can also affect cell counts. The drop of blood on the slide contains millions of RBCs, thousands of WBCs, and hundreds of thousands of platelets. Under the microscope, the stained WBCs can be easily seen and counted to estimate the number of each type of cell present. In addition, one can compare their size, shape, and general appearance to the established appearance of normal cells. It is possible to distinguish between the five different types of WBCs and to determine their relative percentages by counting 100 consecutive cells. During this examination, one can also evaluate the size, shape, and colour of the RBCs and also estimate the number of platelets present. White blood cells have a nucleus surrounded by cytoplasm. In the bone marrow, they differentiate into two groups: myelocytic and lymphoid cells. They then mature into five distinct types of WBCs.

Neutrophils cells that have cytoplasm with pink or purple granules. They compose the majority of WBCs in a healthy adult. Eosinophils - are easily recognized in stained smears with their large, red-orange granules. Generally low in number (1-3%), most often become elevated in number in individuals with allergies and parasitic infections.

Basophils - all have large, black granules and are the least often seen type of WBC (1%). Increased numbers of basophiles are not often encountered but may be elevated in certain leukemias, chicken pox, ulcerative colitis, or after an immunization.

Monocytes - are usually the largest of the WBCs (12-20 m) and are often referred to as scavenger cells (phagocytes). They can ingest particles such as cellular debris, bacteria, or other insoluble particles.

NURSYAZMIN BINTI KHAIRUDDIN (12273208031) MEDICAL IMMUNOLOGY (HCB 21303)

Lymphoid cells lymphocytes are smaller in size (10-12 m) and have a homogeneous cytoplasm and a smooth, round nucleus. These cells are responsible for the production of antibodies (immunoglobulins). When the blood was mixed with the Candida albican, the reaction of the white blood cells and the Candida albican that was obtained showed that the white blood cells was engulfing the Candida albican while some white blood cells already had Candida albican inside the cells. This was because the white blood cells were reacting with the foreign materials, Candida albican, that enter the blood. They engulfing the Candida albican and destroying or eat it. Conclusion: As a conclusion, human white blood cells will react with any foreign materials that enter the blood stream because it is a part of human body immune system. If human was being infected with any microorganisms, the physician will diagnose the infected blood using the blood smear and blood film in order to identify the types of infection because blood smear and blood films results mostly are accurate and it also cheap test.