BLOOD FILM MAKING (THICK AND THIN FILM)
THICK FILM; Thick blood film is carried out to detect the presence or absence of parasite in a given sample.
APPROACH
TITLE: Blood examination
GIVEN: slide, blood sample D, dropper, spreader absorbent cotton wool Giemsa`s stain, staining rack, buffer solution pH 7.0, microscope, immersion oil
PROCE DURE:
A drop blood sample D was place at the center of a slide
A Spreader was used to spread the blood in circular form
It was allowed to air dry.
PROCEDURE FOR STAINING THICK FILM
The dried film was placed on a staining rack
It was covered with 1:10 Giemsa`s stain
It was allowed to stain for 60 minutes
It was washed with buffer solution pH7.0
The back of the stained film was cleaned with absorbent cotton wool
It was allowed to air dry
It was examined microscopically using x100 objective
RESULT:
Plasmodium species seen
Trypanosoma species seen
PROCEDURE FOR MAKING THIN FILM
A drop of sample D was placed at one end of a slide
A spreader was placed just in front and draw back to touch the blood
It was allowed to flow through the edges of the spreader
It was pushed forward gently to the other end
It was allowed to air dry
STAINING OF THIN BLOOD FILM
TITLE: Blood examination
GIVEN: Lieshman`s stain, dried thin film, staining rack buffer solution pH6.8 cotton wool immersion oil
PROCEDURE: The dried thin film was placed on a staining rack
It was covered with lieshman`s stain for 2minutes
It was diluted with buffer solution PH6.8 for 8minutes
It was washed with buffer solution PH6.8
The back of the stained film was cleaned with absorbent cotton wool
It was allowed to air dry
It was examined microscopically using x100
RESULT:
Plasmodium specie seen
Trypanosome specie seen
Lieshmaina specie seen
EXAMINATION OF URINE SAMPLE
This is the parasitological examination of freshly voided urine and the documentation of its clinical significance in a given sample, this aspect gives the clinical feature of urine specime.
METHOD
MICROSCOPY
Question process urine sample H and report for findings
TITLE: Urine microscopy
GIVEN: Urine sample marked H, microscope, centrifuge, slide, cover slip, centrifuge tube
AIM: To process urine sample H and report for parasite
PROCEDURE:
10ml of well mixed urine sample H was placed in a centrifuge tube
It was centrifuged at 15000rpm for 5minutes
Supernatant was decanted
The deposit was re-suspended
A drop of well mixed urine deposit was place at the center of a slide
It was covered with cover slip
It was examined systematically on the microscope using x10 objective and was confirmed with x40 objective
RESULT:
Ovum of schistosoma haematobium seen
Trichomonas vaginalis seen
ASSIGNMENT: List all the parasite that could be found in urine and draw two of the parasite
URINE DEPOSITE EXAMINATION
Question: Examine the urine deposit and record your findings
APPROUCH TO PROFESSIONAL EXAMINATION
TITLE: Urine microscopy
GIVEN: Urine deposit, slide, cover slip, microscope
AIM: To examine urine deposit and record for findings
PROCEDURE: Urine deposit was re-suspended
A drop was place at the centre of the slide
It was covered with cover slip
It was examine microscopically using x10 and confirm with x40 objective respectively
RESULT
Ovum of Schistosoma haematobium seen
Epithelial cell seen
STOOL EXAMINATION
This laboratory diagnostic procedure is use for the detection of parasite in stool sample.
QUESTION: Examine stool sample A for the presences of parasite
TITLE: Stool microscopy
GIVEN: Normal saline, Lugol`s iodine, Microscope, Microscopic slide, applicator stick, Stool sample A, cover slide.
AIM: To examine stool sample A for the presence of parasites
PROCEDURE: A drop of normal saline was placed at one end of the slide and Lugol`s iodine at the other end.
Applicator stick was used to obtain a small portion of the stool sample .
It was emulsified in a drop of normal saline and Lugol`s iodine respectively.
Both smears were covered with cover slip.
Both preparations were examined systematically with microscope using x10 objective and x40 objective respectively.
RESULT
Ovum of hookworm seen
Ascaris lumbricoides seen
Entamoeba histolytica seen
Enterobius vermicularis seen
EXAMINATION OF BLOOD
PROFESSIONAL EXAMINATION APPROACH
Question: Examine blood sample H for presence of parasite
Wet preparation Method
TITLE: Blood examination
GIVEN; Blood sample H, normal saline ,slide ,cover slip ,microscope
AMI: To examine blood sample H the presence of parasite
PROCEDURE:
-A drop of normal saline was placed at the center of a slide
-A drop of a well mixed blood was added
-It was mixed and covered with cover slip
-It was examined microscopically using x10 objective and confirm with x40 objective respectively.
RESULT
Wuchereria bancrofti seen
Loa loa seen
Brugia malayi seen
Onchocerca voluvalus seen
Mangonella ozar seen
HAEMATOLOGY
PACKED CELL VOLUME (PCV)
PCV: means the volume of red blood cells concentration as compared to the volume of whole blood.
VALUE OF TEST: This is used to screen for anaemia and to diagnose polycythaemia and to monitor its treatment.
PROFESSIONAL EXAMINATION APPROUCH
Carry out packed cell volume on sample k and report your findings state the reference values for male, female and infant
TITLE: packed cell volume estimation
GIVEN: Blood sample k, sealing clay, haematocrite reader haematocrit centrifuge cotton wool
AIM: To carry out packed cell volume on sample k and report for findings
PRINCIPLE: when whole blood is centrifuged in a specialize tube at 15,000rpm for 5minutes the red cells sediment at the bottom followed by layer of buffy coat and then plasma .The relative mass of red cells is been estimated using haematocrit reader.
Procedure:
The capillary tube was filled to ¾ with well mixed blood sample K
One end was sealed with sealing clay
It was centrifuged at 15,000 rpm for 5 minutes
The result was read using haematocrit reader and recorded
REFERENCE VALUES:
Male 40-50
Female 36-46
Infant 35-54
HAEMOGLOBIN ESTIMATION
Hemoglobin is the main protein present in red blood cells. Its functions include; transportation and distribution of oxygen content. it also remove some waste products like carbon dioxide from the tissues. Since hemoglobin is concern with the distribution of oxygen, hemoglobin estimation is one of the most frequently requested tests in hematology laboratory. There are various methods for hemoglobin estimation. These include:
1. Cyanmethaemoglobin method
2. Alkaline haematin method
3. Oxyhaemoglobin method
4. Oxygen-carrying capacity of blood
5. Sahli`s method or acid haematin method
6. Copper sulphate method
Out the method mentioned above our emphasize on cyanmethaemoglobin and sahli`s.
APPROACH
TITLE: Hemoglobin estimation
GIVEN: Drab kin’s solution, 0.02 ml pipette, test tubes, test tube rack, colorimeter, sample k
AIM: To carry out hemoglobin on sample K
PROCEDURE:
5ml of drabkin`s solution was placed in a clean dry test tube
0.02ml of blood sample K was added
It was mixed and allowed to stand for 15minutes
The standard was treated same as test
Both were read at 540nm using a colorimeter and recorded
PRINCIPLE: Haemoglobin is converted to methaemoglobin by the action of potassium ferricyanide, the methaemoglobin is in turn converted to cyanmethaemoglobin by the action of potassium cyanide. The color intensity of the solution is directly proportional to the amount of Haemoglobin present in a sample of whole blood.
RESULT
O.D of blood sample k -0.3
O.D of std -0.4
Diluting factor -251
Conc.of std 60g/dl
Calculation
Hb con.= O.D of test xcon. Of std x diluting factor
O.D of stdx1000
= 0.3x60x251
0.4x1000
= 4518
400
=11.3g/dl
SAHLI`S METHOD
TITLE: Hemoglobin estimation
GIVEN: Blood sample, Hb pipette, hemometer, sahli`s graduated tube , n/10HCL
AIM: To estimate hemoglobin concentration of blood sample k
PRINCIPLE: Hemoglobin is converted to acid hematin by normal 10 hydrochloric acid to a coloured compound which is compared with a standard to estimate hemoglobin concentration
PROCEDURE: N/10hcl was filled in sahli`s graduated tube to mark 20 ,0.02ml of well mixed blood sample k was added it was mixed and allowed to stand for 5munites it was compared with standard it was diluted with n/10hcl drop by drop and mixed at each interval and compared with standard until the colour matches with standard the result was read in g/dl and recorded
RESULT
Hb Con.=14.50g/dl
Reference Value:
Children at birth: 13.5-19.5g/dl
Male:13.0-18.0g/dl
Female:12.0-15.og/dl
BLOOD GROUPING
APPROACH
QUESTION:carry out cell and rhesus grouping on sample M,N,K and L and report for the sample that has blood group A,B,AB and o
TITLE: Cell grouping
GIVEN: Sample M,N,K and l Anti A,B,AB and 0, tile, A cells B cells o cells and applicator stick
AIM: To carry out cell and rhesus grouping on sample M,N,K and L and report the sample has blood group A ,B,AB and o
PROCEDURE:
A drop of sample M,N,K and L was placed at a tile in three depression
A drop of Anti A,B,AB and O was added respectively mixed
It was rocked for 5minute and observed for agglutination
PROTOCOL TABLE
Anti-A
Anti-B
Ant-AB
Anti-D
Blood group
controls
A cells
+
-
+
-
B cells
-
+
+
-
O cells
-
-
-
-
samples
M
+
-
+
-
A-
N
-
+
+
+
B+
K
-
-
-
+
O+
L
+
+
+
-
AB-
KEY: + agglutination
-No agglutination
RESULT
Sample M: has blood group A rh D-
Sample N: has blood group B rh D+
Sample K: has blood group O rh D+
Sample L: has blood group AB rh D-
BACTERIOLOGY
LABORATORY CULTUR MEDIA
This are artificial medium used for growing of micro-organisms in bacteriology laboratory, these media allow micro-organisms to grow as if their in the body system, contain all nutritional requirement that allow for the growth of micro-organisms.
EXAMPLES OF MEDIA
1. Basal media; this are sample laboratory media used for the growth of most micro-organisms. they contain the basic nutrients: peptone ,minerals salts, and water: the media do not require special nutritional requirement. Example are nutrient broth, infusion broth etc
2. Enriched media. These are solide media used for the isolation of delicate organism that would not grow on basal media except enriching factor such as blood, serum, or egg is added. Examples are blood, serum and chocolate agars.
3. Enrichment media. These is liquid selective media consisting of substances that will inhibit the growth of some organism and allow the growth of some organisms e.g. selenite Fbroth inhibits the growth of coliform bacilli and promote the growth that of typhoid and paratyphoid organisms.
4. Selective media. These are solid media that promote the growth of one organism and inhibit the growth of other organisms e.g. DCA (desoxychocolate citrate agar) support the growth of salmonella and shigella groups. Bismuth sulphite agar promotes the growth, isolation and differentiation of typhoid bacilli from the other fecal bacteria normally present in stool.
ASSIGNMENT
1. Name laboratory media and their nutritional requirement
2. State the guidelines involve in preparing laboratory media.
3. State one usefulness Gram staining apart from the ones on this booklet
4. Elaborate the principle of Gram`s staining
AGAR
Agar-agar or agar is a carbohydrate (poly saccharide) derive from certain seaweeds.it contains inorganic salts,protein-like minerals (magnesium and calcium) it is a solidifying agent it best suitable at approximately 1% concentration it is a good solidifying agent available in powder form.
CULTURE
A microbiological culture or microbial culture is a method of multiplying microbial organisms by lettering them reproduce in a predetermined culture medium under controlled laboratory conditions or is the process of growing a bacterial or other biological entity in an artificial medium.
COLONY
This is seen as a visible mass of micro-organisms all originating from a single mother or cell, therefore a colony constitutes a clone of bacteria all genetically alike or their several individual organisms especially of thre same species living together in a close association.(cell culture).it can also be seen as a cluster of identical cells (clones) on the surface a solide media.
PREPARATION AND POURING OF PLATS
QUESTION; i. prepare and pour the following agar plats
Nutrient agar, blood agar and chocolate agar(10%) sub-culture sample j for discrete colonies and stain by Gram`s staining technique
ii. What is the usefulness of this staining method?
APPROACH
TITLE: Pouring of nutrient agar
GIVEN: Molten nutrient agar, sterile Petri dish
AIM: To prepare and pour nutrient agar plate
PROCEDURE: 20ml of molten nutrient agar was allowed to cool to 450 it was poured asceptically into a sterile Petri dish it was covered and allowed to solidified
POURING OF 10% BLOOD AGAR PLATE
TITLE: pouring of blood agar plate
GIVEN: molten nutrient agar, blood sample, sterile Petri dish
AIM: To prepare and pour 10% blood agar plate
PROCEDURE: 20ml of molten nutrient agar was allowed to cool to 450 c 2ml of molten nutrient agar was removed aseptically based on the calculation below:
RV
O where: R =required concentration=10%
V=final volume =20ml
O=original concentration=100%
10x20 =2ml
100
It was mixed and poured ascetically in to a sterile Petri dish it was allow to solidify.
POUERING OF CHOCOLATE
TITLE: pouring of chocolate
GIVEN: molten nutrient agar, blood sample, bunsenbuner, sterile Petri dish
AIM: To prepare and pour chocolate agar plate
PROCEDURE: 20ml of molten nutrient agar was allow to cool to 450 c 2ml molten nutrient agar was removed ascetically based on the calculation below:
RV
O
Where: R= required concentration =10%
V= final volume =20ml
O= original concentration =100%
10%X20ml =2ml
100%
It was mixed and reheated till colour turns chocolate it was allowed to cool to 450 c it was poured aseptically into sterile Petri dish it was covered and allowed to solidify.
INOCULATION (STREAKING)
TITLE: Inoculation (streaking)
GIVEN: Broth culture, wire loop, Bunsen burner,plate
AIM: To inoculate sample h on culture plate
PROCEDURE: The solidified plate was dried in the incubator at 370 c
wire loop was sterilized and allowed to cool
A loopful of a broth culture was obtained and a pool was made at area marked A as shown below the wire loop was re-sterilized and allowed to cool and streaked from area A to area B as shown below same technique was repeated to area C,D,E and F as shown below
GRAM`S STAINING
This staining technique differentiate bacteria into two group Gram positive and Gram negative organisms as propound my Christian Gram in 1884.
MAKING SMEAR WITH SOLID CULTURE
TITLE: Smear preparation
GIVEN: Sample H, wire loop, Bunsen burner ,normal saline,slide , applicator stick
AIM: To prepare a smear from solid culture
PROCEDURE: A drop of normal saline was placed at the center of a slide a wire loop was sterilized and allowed to cool a colony of sample H was picked it was emulsified in the drop of normal saline it was allowed to air dry
TITLE: Gram`s staining method
GIVEN: crystal violet, Gram`s iodine, acetone, neutral red, Bunsen burner, dried smear, staining rack distilled water
AIM: To stain sample H by Gram`s staining method
PROCEDURE: The dried smear was passed through Bunsen flame three times and was allowed to cool it was placed on a staining rack it was covered with crystal violet for 1minute it was wash with was water it was covered with Gram`s iodine for 1minute it was wash with water it was decolorized with acetone for 30second it was wash with water it was covered with neutral red for 1minute it was wash with water the back of the slide was cleaned with absorbent cotton wool it was allowed to air dry it was examined microscopically using x100 objective
RESULT:
Gram positive cocci seen (Blue)
Gram`s negative rods seen (red)
ii. Usefulness of the method
1.It differentiate bacteria into Gram positive and Gram negative groups
2. It demonstrates the typical arrangement of bacteria as chains, clusters or chine’s lettering
3.It shows the morphology of bacteria as coccobacilli etc
4. It gives the choice of the antibiotic disc for sensitivity.
PREPARATION OF STAINS
Gram`s staining reagents:
Crystal violet
Constituents
Crystal violet -0.5g
Distilled water -100ml
Weigh crystal violet powder and dissolve in distilled water mix well, label filter before use
Gram`s iodine (mordant)
Constituents
Iodine -1g
Potassium iodine -2g
Distilled water -300ml
Preparation: dissolve potassium iodine in distilled water, add the iodine crystals, dissolve by shaking , mix well label and store in a tightly stoppedred bottle . filter before use.
DECOLOURISER (ABSOLUTE ALCOHOL, ACETONE)
Alcohol and acetone are commercially purchased
COUNTER STIAN (SAFRANIN, NEUTRAL RED, DILUTE CARBOL FUCHSIN)
SAFRANIN
Constituents
Safranin -5g
Distilled water -1000ml
Preparation
Dissolve thez dye in the distilled water , mix well, filter , put in bottle label with date and store.
NEUTRAL RED
Constituents
Neutral red -1g
Acetic acid -2ml
Distilled water -1000ml
Preparation
Dissolve the dye in distilled water , add the acid mix well filter before use put in label bottles with date and store.
DILUTE CARBOL FUCHSIN
Constituents
Strong acid fuchsin -10ml
Distilled water -90ml
Preparation
Measure the constituents mix together , filter put in label bottles with date and store
PREPARATION OF STAINS AND REAGENT USE IN PARASITOLOGY AND HAEMATOLOGY LABORATORY
EOSIN
Constituents
Eosin -1g
Distilled water -100ml
Thymol (preservative) -1crystal
Preparation
Weigh eosin powder, dissolve in distilled water, finally add thymol crystal, mix well put in label bottle and store.
NORMAL SALIN (PHYSIOLOGICAL SALIN OR ISOTONIC SALIN)
Constituents
Sodium chloride -0.8g
Distilled water -100ml
Preparation
Weigh sodium chloride and dissolve in distilled water mix well put in bottles label with date
GIEMSA`S STAIN
Constituents
Azure II eosin -3g
Azure II -0.8g
Glycerol -200ml
Absolute alcohol -300ml
Preparation
Grand the two dyes weigh each powder accurately measure alcohol and glycerol and mix together dissolve both powder in the mixture and place in a water bath at 50-600 c for two hours to finally dissolve shake gently at half-our intervals. The stain should stand at room temperature for three weeks in dark brown bottles with label and date and should be filter before use.
GIEMSA`S STAIN BUFFER SOLUTION pH7.0
Constituents
Anhydrous disodium hydrogen phosphate -0.58g
Anhydrous potassium dihydrogen phosphate -0.35g
Distilled water -1000ml
Preparation
Dissolve the phosphate in distilled water; check that the pH of the resultant buffer is 7.0 put in bottles label with date and store.
LIESHMAN`S STAIN pH 6.8
Constituents
Lieshman`s stain- 0.15g
Absolute alcohol -100ml
Preparation
Dissolve the stain in alcohol, plug the neck with cotton wool and warm gently in water bath for 15minutes, shake at intervals. Store in a dark tightly stoppered bottle and allow to stand for 24hours before use.
BUFFER SOLUTION pH6.8
Constituents
Anhydrous disodium phosphate 0.47g
Anhydrous potassium dihydrogen phosphate o.46g
Distilled water -1000ml
Preparation
Dissolve the phosphate in the distelled water;check that the pH of the resultant buffer is 6.8 put in tightly dark label bottles with date and store.
CLINICAL PATHOLOGY
URINE ANALYSIS:
This is defined as the physical, chemical and microscopic examination of urine in other word called routine urine analysis. Urine analysis in an important part of the initial examination of a patient and the results provide a valuable picture of the patient’s general health Pattern.
physical
chemical
microscopy
colour(amber)
pH
red blood cells
Transparency(clear)
Glucose
white blood cells
volume(1-2litres)
ketone
cast
odour(aromatic)
protein
epithelial cells
bilirubin
bacteria
urobilinogen
crystals
blood
e.t.c.
Note: Nitrite, ascorbic acid specific gravity and HCG can also be detected, using commercial available strips which include:
.Combi 9, 10, 11 etc
.Pregnancy strip test (pt).
Method :Dipstick test or or urine test strip
Value of test: for detection of diabetes, metabolic abnormalities, liver, disease, hemolytic disease, and kidney and urinary tract diseases.
Question carry out chemical urine analysis on sample h and report for findings.
STRIP METHOD FOR URINANALYSIS
TITLE: chemical urinalysis
GIVEN: Urine sample h, combi 9 strip
AIM: To carry out chemical urinalysis on sample h and report for findings
PRINCIPLE: in the presence of glucose oxidase, glucose is oxidized to gluconic acid and hydrogen peroxide (glucose oxidase is specific for glucose) glucose+02 +H20 gluconic acid in the presence of peroxides, hydrogen peroxide will oxidize a colorless chromogen, an oxygen acceptor, to a coloured complex.
PROCEDURE: as show in the protocol table below
METHOD
OBSERVATION
INFERENCE
A combi 9 strip was removed and the container was closed back it was dipped in urine sample h it was removed and the exess urine was drained it was allowed for 60 seconds it was compared with colour chart.
Protein: colour change
Bilirubin: colour change
Ketones: colour change
protein present
bilirubin present
ketone present
THICK FILM; Thick blood film is carried out to detect the presence or absence of parasite in a given sample.
- Question: you are given a blood sample D, Make thick and thin blood film and stain by Giemsa’s Staining Method.
APPROACH
TITLE: Blood examination
GIVEN: slide, blood sample D, dropper, spreader absorbent cotton wool Giemsa`s stain, staining rack, buffer solution pH 7.0, microscope, immersion oil
PROCE DURE:
A drop blood sample D was place at the center of a slide
A Spreader was used to spread the blood in circular form
It was allowed to air dry.
PROCEDURE FOR STAINING THICK FILM
The dried film was placed on a staining rack
It was covered with 1:10 Giemsa`s stain
It was allowed to stain for 60 minutes
It was washed with buffer solution pH7.0
The back of the stained film was cleaned with absorbent cotton wool
It was allowed to air dry
It was examined microscopically using x100 objective
RESULT:
Plasmodium species seen
Trypanosoma species seen
PROCEDURE FOR MAKING THIN FILM
A drop of sample D was placed at one end of a slide
A spreader was placed just in front and draw back to touch the blood
It was allowed to flow through the edges of the spreader
It was pushed forward gently to the other end
It was allowed to air dry
STAINING OF THIN BLOOD FILM
TITLE: Blood examination
GIVEN: Lieshman`s stain, dried thin film, staining rack buffer solution pH6.8 cotton wool immersion oil
PROCEDURE: The dried thin film was placed on a staining rack
It was covered with lieshman`s stain for 2minutes
It was diluted with buffer solution PH6.8 for 8minutes
It was washed with buffer solution PH6.8
The back of the stained film was cleaned with absorbent cotton wool
It was allowed to air dry
It was examined microscopically using x100
RESULT:
Plasmodium specie seen
Trypanosome specie seen
Lieshmaina specie seen
EXAMINATION OF URINE SAMPLE
This is the parasitological examination of freshly voided urine and the documentation of its clinical significance in a given sample, this aspect gives the clinical feature of urine specime.
METHOD
MICROSCOPY
Question process urine sample H and report for findings
TITLE: Urine microscopy
GIVEN: Urine sample marked H, microscope, centrifuge, slide, cover slip, centrifuge tube
AIM: To process urine sample H and report for parasite
PROCEDURE:
10ml of well mixed urine sample H was placed in a centrifuge tube
It was centrifuged at 15000rpm for 5minutes
Supernatant was decanted
The deposit was re-suspended
A drop of well mixed urine deposit was place at the center of a slide
It was covered with cover slip
It was examined systematically on the microscope using x10 objective and was confirmed with x40 objective
RESULT:
Ovum of schistosoma haematobium seen
Trichomonas vaginalis seen
ASSIGNMENT: List all the parasite that could be found in urine and draw two of the parasite
URINE DEPOSITE EXAMINATION
Question: Examine the urine deposit and record your findings
APPROUCH TO PROFESSIONAL EXAMINATION
TITLE: Urine microscopy
GIVEN: Urine deposit, slide, cover slip, microscope
AIM: To examine urine deposit and record for findings
PROCEDURE: Urine deposit was re-suspended
A drop was place at the centre of the slide
It was covered with cover slip
It was examine microscopically using x10 and confirm with x40 objective respectively
RESULT
Ovum of Schistosoma haematobium seen
Epithelial cell seen
STOOL EXAMINATION
This laboratory diagnostic procedure is use for the detection of parasite in stool sample.
QUESTION: Examine stool sample A for the presences of parasite
TITLE: Stool microscopy
GIVEN: Normal saline, Lugol`s iodine, Microscope, Microscopic slide, applicator stick, Stool sample A, cover slide.
AIM: To examine stool sample A for the presence of parasites
PROCEDURE: A drop of normal saline was placed at one end of the slide and Lugol`s iodine at the other end.
Applicator stick was used to obtain a small portion of the stool sample .
It was emulsified in a drop of normal saline and Lugol`s iodine respectively.
Both smears were covered with cover slip.
Both preparations were examined systematically with microscope using x10 objective and x40 objective respectively.
RESULT
Ovum of hookworm seen
Ascaris lumbricoides seen
Entamoeba histolytica seen
Enterobius vermicularis seen
EXAMINATION OF BLOOD
PROFESSIONAL EXAMINATION APPROACH
Question: Examine blood sample H for presence of parasite
Wet preparation Method
TITLE: Blood examination
GIVEN; Blood sample H, normal saline ,slide ,cover slip ,microscope
AMI: To examine blood sample H the presence of parasite
PROCEDURE:
-A drop of normal saline was placed at the center of a slide
-A drop of a well mixed blood was added
-It was mixed and covered with cover slip
-It was examined microscopically using x10 objective and confirm with x40 objective respectively.
RESULT
Wuchereria bancrofti seen
Loa loa seen
Brugia malayi seen
Onchocerca voluvalus seen
Mangonella ozar seen
HAEMATOLOGY
PACKED CELL VOLUME (PCV)
PCV: means the volume of red blood cells concentration as compared to the volume of whole blood.
VALUE OF TEST: This is used to screen for anaemia and to diagnose polycythaemia and to monitor its treatment.
PROFESSIONAL EXAMINATION APPROUCH
Carry out packed cell volume on sample k and report your findings state the reference values for male, female and infant
TITLE: packed cell volume estimation
GIVEN: Blood sample k, sealing clay, haematocrite reader haematocrit centrifuge cotton wool
AIM: To carry out packed cell volume on sample k and report for findings
PRINCIPLE: when whole blood is centrifuged in a specialize tube at 15,000rpm for 5minutes the red cells sediment at the bottom followed by layer of buffy coat and then plasma .The relative mass of red cells is been estimated using haematocrit reader.
Procedure:
The capillary tube was filled to ¾ with well mixed blood sample K
One end was sealed with sealing clay
It was centrifuged at 15,000 rpm for 5 minutes
The result was read using haematocrit reader and recorded
REFERENCE VALUES:
Male 40-50
Female 36-46
Infant 35-54
HAEMOGLOBIN ESTIMATION
Hemoglobin is the main protein present in red blood cells. Its functions include; transportation and distribution of oxygen content. it also remove some waste products like carbon dioxide from the tissues. Since hemoglobin is concern with the distribution of oxygen, hemoglobin estimation is one of the most frequently requested tests in hematology laboratory. There are various methods for hemoglobin estimation. These include:
1. Cyanmethaemoglobin method
2. Alkaline haematin method
3. Oxyhaemoglobin method
4. Oxygen-carrying capacity of blood
5. Sahli`s method or acid haematin method
6. Copper sulphate method
Out the method mentioned above our emphasize on cyanmethaemoglobin and sahli`s.
APPROACH
TITLE: Hemoglobin estimation
GIVEN: Drab kin’s solution, 0.02 ml pipette, test tubes, test tube rack, colorimeter, sample k
AIM: To carry out hemoglobin on sample K
PROCEDURE:
5ml of drabkin`s solution was placed in a clean dry test tube
0.02ml of blood sample K was added
It was mixed and allowed to stand for 15minutes
The standard was treated same as test
Both were read at 540nm using a colorimeter and recorded
PRINCIPLE: Haemoglobin is converted to methaemoglobin by the action of potassium ferricyanide, the methaemoglobin is in turn converted to cyanmethaemoglobin by the action of potassium cyanide. The color intensity of the solution is directly proportional to the amount of Haemoglobin present in a sample of whole blood.
RESULT
O.D of blood sample k -0.3
O.D of std -0.4
Diluting factor -251
Conc.of std 60g/dl
Calculation
Hb con.= O.D of test xcon. Of std x diluting factor
O.D of stdx1000
= 0.3x60x251
0.4x1000
= 4518
400
=11.3g/dl
SAHLI`S METHOD
TITLE: Hemoglobin estimation
GIVEN: Blood sample, Hb pipette, hemometer, sahli`s graduated tube , n/10HCL
AIM: To estimate hemoglobin concentration of blood sample k
PRINCIPLE: Hemoglobin is converted to acid hematin by normal 10 hydrochloric acid to a coloured compound which is compared with a standard to estimate hemoglobin concentration
PROCEDURE: N/10hcl was filled in sahli`s graduated tube to mark 20 ,0.02ml of well mixed blood sample k was added it was mixed and allowed to stand for 5munites it was compared with standard it was diluted with n/10hcl drop by drop and mixed at each interval and compared with standard until the colour matches with standard the result was read in g/dl and recorded
RESULT
Hb Con.=14.50g/dl
Reference Value:
Children at birth: 13.5-19.5g/dl
Male:13.0-18.0g/dl
Female:12.0-15.og/dl
BLOOD GROUPING
APPROACH
QUESTION:carry out cell and rhesus grouping on sample M,N,K and L and report for the sample that has blood group A,B,AB and o
TITLE: Cell grouping
GIVEN: Sample M,N,K and l Anti A,B,AB and 0, tile, A cells B cells o cells and applicator stick
AIM: To carry out cell and rhesus grouping on sample M,N,K and L and report the sample has blood group A ,B,AB and o
PROCEDURE:
A drop of sample M,N,K and L was placed at a tile in three depression
A drop of Anti A,B,AB and O was added respectively mixed
It was rocked for 5minute and observed for agglutination
PROTOCOL TABLE
Anti-A
Anti-B
Ant-AB
Anti-D
Blood group
controls
A cells
+
-
+
-
B cells
-
+
+
-
O cells
-
-
-
-
samples
M
+
-
+
-
A-
N
-
+
+
+
B+
K
-
-
-
+
O+
L
+
+
+
-
AB-
KEY: + agglutination
-No agglutination
RESULT
Sample M: has blood group A rh D-
Sample N: has blood group B rh D+
Sample K: has blood group O rh D+
Sample L: has blood group AB rh D-
BACTERIOLOGY
LABORATORY CULTUR MEDIA
This are artificial medium used for growing of micro-organisms in bacteriology laboratory, these media allow micro-organisms to grow as if their in the body system, contain all nutritional requirement that allow for the growth of micro-organisms.
EXAMPLES OF MEDIA
1. Basal media; this are sample laboratory media used for the growth of most micro-organisms. they contain the basic nutrients: peptone ,minerals salts, and water: the media do not require special nutritional requirement. Example are nutrient broth, infusion broth etc
2. Enriched media. These are solide media used for the isolation of delicate organism that would not grow on basal media except enriching factor such as blood, serum, or egg is added. Examples are blood, serum and chocolate agars.
3. Enrichment media. These is liquid selective media consisting of substances that will inhibit the growth of some organism and allow the growth of some organisms e.g. selenite Fbroth inhibits the growth of coliform bacilli and promote the growth that of typhoid and paratyphoid organisms.
4. Selective media. These are solid media that promote the growth of one organism and inhibit the growth of other organisms e.g. DCA (desoxychocolate citrate agar) support the growth of salmonella and shigella groups. Bismuth sulphite agar promotes the growth, isolation and differentiation of typhoid bacilli from the other fecal bacteria normally present in stool.
ASSIGNMENT
1. Name laboratory media and their nutritional requirement
2. State the guidelines involve in preparing laboratory media.
3. State one usefulness Gram staining apart from the ones on this booklet
4. Elaborate the principle of Gram`s staining
AGAR
Agar-agar or agar is a carbohydrate (poly saccharide) derive from certain seaweeds.it contains inorganic salts,protein-like minerals (magnesium and calcium) it is a solidifying agent it best suitable at approximately 1% concentration it is a good solidifying agent available in powder form.
CULTURE
A microbiological culture or microbial culture is a method of multiplying microbial organisms by lettering them reproduce in a predetermined culture medium under controlled laboratory conditions or is the process of growing a bacterial or other biological entity in an artificial medium.
COLONY
This is seen as a visible mass of micro-organisms all originating from a single mother or cell, therefore a colony constitutes a clone of bacteria all genetically alike or their several individual organisms especially of thre same species living together in a close association.(cell culture).it can also be seen as a cluster of identical cells (clones) on the surface a solide media.
PREPARATION AND POURING OF PLATS
QUESTION; i. prepare and pour the following agar plats
Nutrient agar, blood agar and chocolate agar(10%) sub-culture sample j for discrete colonies and stain by Gram`s staining technique
ii. What is the usefulness of this staining method?
APPROACH
TITLE: Pouring of nutrient agar
GIVEN: Molten nutrient agar, sterile Petri dish
AIM: To prepare and pour nutrient agar plate
PROCEDURE: 20ml of molten nutrient agar was allowed to cool to 450 it was poured asceptically into a sterile Petri dish it was covered and allowed to solidified
POURING OF 10% BLOOD AGAR PLATE
TITLE: pouring of blood agar plate
GIVEN: molten nutrient agar, blood sample, sterile Petri dish
AIM: To prepare and pour 10% blood agar plate
PROCEDURE: 20ml of molten nutrient agar was allowed to cool to 450 c 2ml of molten nutrient agar was removed aseptically based on the calculation below:
RV
O where: R =required concentration=10%
V=final volume =20ml
O=original concentration=100%
10x20 =2ml
100
It was mixed and poured ascetically in to a sterile Petri dish it was allow to solidify.
POUERING OF CHOCOLATE
TITLE: pouring of chocolate
GIVEN: molten nutrient agar, blood sample, bunsenbuner, sterile Petri dish
AIM: To prepare and pour chocolate agar plate
PROCEDURE: 20ml of molten nutrient agar was allow to cool to 450 c 2ml molten nutrient agar was removed ascetically based on the calculation below:
RV
O
Where: R= required concentration =10%
V= final volume =20ml
O= original concentration =100%
10%X20ml =2ml
100%
It was mixed and reheated till colour turns chocolate it was allowed to cool to 450 c it was poured aseptically into sterile Petri dish it was covered and allowed to solidify.
INOCULATION (STREAKING)
TITLE: Inoculation (streaking)
GIVEN: Broth culture, wire loop, Bunsen burner,plate
AIM: To inoculate sample h on culture plate
PROCEDURE: The solidified plate was dried in the incubator at 370 c
wire loop was sterilized and allowed to cool
A loopful of a broth culture was obtained and a pool was made at area marked A as shown below the wire loop was re-sterilized and allowed to cool and streaked from area A to area B as shown below same technique was repeated to area C,D,E and F as shown below
GRAM`S STAINING
This staining technique differentiate bacteria into two group Gram positive and Gram negative organisms as propound my Christian Gram in 1884.
MAKING SMEAR WITH SOLID CULTURE
TITLE: Smear preparation
GIVEN: Sample H, wire loop, Bunsen burner ,normal saline,slide , applicator stick
AIM: To prepare a smear from solid culture
PROCEDURE: A drop of normal saline was placed at the center of a slide a wire loop was sterilized and allowed to cool a colony of sample H was picked it was emulsified in the drop of normal saline it was allowed to air dry
TITLE: Gram`s staining method
GIVEN: crystal violet, Gram`s iodine, acetone, neutral red, Bunsen burner, dried smear, staining rack distilled water
AIM: To stain sample H by Gram`s staining method
PROCEDURE: The dried smear was passed through Bunsen flame three times and was allowed to cool it was placed on a staining rack it was covered with crystal violet for 1minute it was wash with was water it was covered with Gram`s iodine for 1minute it was wash with water it was decolorized with acetone for 30second it was wash with water it was covered with neutral red for 1minute it was wash with water the back of the slide was cleaned with absorbent cotton wool it was allowed to air dry it was examined microscopically using x100 objective
RESULT:
Gram positive cocci seen (Blue)
Gram`s negative rods seen (red)
ii. Usefulness of the method
1.It differentiate bacteria into Gram positive and Gram negative groups
2. It demonstrates the typical arrangement of bacteria as chains, clusters or chine’s lettering
3.It shows the morphology of bacteria as coccobacilli etc
4. It gives the choice of the antibiotic disc for sensitivity.
PREPARATION OF STAINS
Gram`s staining reagents:
Crystal violet
Constituents
Crystal violet -0.5g
Distilled water -100ml
Weigh crystal violet powder and dissolve in distilled water mix well, label filter before use
Gram`s iodine (mordant)
Constituents
Iodine -1g
Potassium iodine -2g
Distilled water -300ml
Preparation: dissolve potassium iodine in distilled water, add the iodine crystals, dissolve by shaking , mix well label and store in a tightly stoppedred bottle . filter before use.
DECOLOURISER (ABSOLUTE ALCOHOL, ACETONE)
Alcohol and acetone are commercially purchased
COUNTER STIAN (SAFRANIN, NEUTRAL RED, DILUTE CARBOL FUCHSIN)
SAFRANIN
Constituents
Safranin -5g
Distilled water -1000ml
Preparation
Dissolve thez dye in the distilled water , mix well, filter , put in bottle label with date and store.
NEUTRAL RED
Constituents
Neutral red -1g
Acetic acid -2ml
Distilled water -1000ml
Preparation
Dissolve the dye in distilled water , add the acid mix well filter before use put in label bottles with date and store.
DILUTE CARBOL FUCHSIN
Constituents
Strong acid fuchsin -10ml
Distilled water -90ml
Preparation
Measure the constituents mix together , filter put in label bottles with date and store
PREPARATION OF STAINS AND REAGENT USE IN PARASITOLOGY AND HAEMATOLOGY LABORATORY
EOSIN
Constituents
Eosin -1g
Distilled water -100ml
Thymol (preservative) -1crystal
Preparation
Weigh eosin powder, dissolve in distilled water, finally add thymol crystal, mix well put in label bottle and store.
NORMAL SALIN (PHYSIOLOGICAL SALIN OR ISOTONIC SALIN)
Constituents
Sodium chloride -0.8g
Distilled water -100ml
Preparation
Weigh sodium chloride and dissolve in distilled water mix well put in bottles label with date
GIEMSA`S STAIN
Constituents
Azure II eosin -3g
Azure II -0.8g
Glycerol -200ml
Absolute alcohol -300ml
Preparation
Grand the two dyes weigh each powder accurately measure alcohol and glycerol and mix together dissolve both powder in the mixture and place in a water bath at 50-600 c for two hours to finally dissolve shake gently at half-our intervals. The stain should stand at room temperature for three weeks in dark brown bottles with label and date and should be filter before use.
GIEMSA`S STAIN BUFFER SOLUTION pH7.0
Constituents
Anhydrous disodium hydrogen phosphate -0.58g
Anhydrous potassium dihydrogen phosphate -0.35g
Distilled water -1000ml
Preparation
Dissolve the phosphate in distilled water; check that the pH of the resultant buffer is 7.0 put in bottles label with date and store.
LIESHMAN`S STAIN pH 6.8
Constituents
Lieshman`s stain- 0.15g
Absolute alcohol -100ml
Preparation
Dissolve the stain in alcohol, plug the neck with cotton wool and warm gently in water bath for 15minutes, shake at intervals. Store in a dark tightly stoppered bottle and allow to stand for 24hours before use.
BUFFER SOLUTION pH6.8
Constituents
Anhydrous disodium phosphate 0.47g
Anhydrous potassium dihydrogen phosphate o.46g
Distilled water -1000ml
Preparation
Dissolve the phosphate in the distelled water;check that the pH of the resultant buffer is 6.8 put in tightly dark label bottles with date and store.
CLINICAL PATHOLOGY
URINE ANALYSIS:
This is defined as the physical, chemical and microscopic examination of urine in other word called routine urine analysis. Urine analysis in an important part of the initial examination of a patient and the results provide a valuable picture of the patient’s general health Pattern.
physical
chemical
microscopy
colour(amber)
pH
red blood cells
Transparency(clear)
Glucose
white blood cells
volume(1-2litres)
ketone
cast
odour(aromatic)
protein
epithelial cells
bilirubin
bacteria
urobilinogen
crystals
blood
e.t.c.
Note: Nitrite, ascorbic acid specific gravity and HCG can also be detected, using commercial available strips which include:
.Combi 9, 10, 11 etc
.Pregnancy strip test (pt).
Method :Dipstick test or or urine test strip
Value of test: for detection of diabetes, metabolic abnormalities, liver, disease, hemolytic disease, and kidney and urinary tract diseases.
Question carry out chemical urine analysis on sample h and report for findings.
STRIP METHOD FOR URINANALYSIS
TITLE: chemical urinalysis
GIVEN: Urine sample h, combi 9 strip
AIM: To carry out chemical urinalysis on sample h and report for findings
PRINCIPLE: in the presence of glucose oxidase, glucose is oxidized to gluconic acid and hydrogen peroxide (glucose oxidase is specific for glucose) glucose+02 +H20 gluconic acid in the presence of peroxides, hydrogen peroxide will oxidize a colorless chromogen, an oxygen acceptor, to a coloured complex.
PROCEDURE: as show in the protocol table below
METHOD
OBSERVATION
INFERENCE
A combi 9 strip was removed and the container was closed back it was dipped in urine sample h it was removed and the exess urine was drained it was allowed for 60 seconds it was compared with colour chart.
Protein: colour change
Bilirubin: colour change
Ketones: colour change
protein present
bilirubin present
ketone present
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