OPD Receiving of samples

➢ Charging of patient laboratory tests 1. Patient/Label Identification – always double

➢ Receiving of samples check the label (full name and date and time
➢ Sample collection (phlebotomy) of collection)
➢ Releasing of results ➢ Urine – ½ to almost full
➢ Stool/feces – pea-sized
➢ Microbiology samples – sterile
Charging of Laboratory test container
➢ AFB – sputum (either 1 or 2 samples
1. Physician’s laboratory request in a clean container with fasting)
2. Fill up patient’s information form ➢ Body Fluids (pleural, synovial,
3. Enter Patient data completely with the name peritoneal, etc.) – depends on the
of the requesting physician and charging laboratory test needed
laboratory request in the system, print charge ➢ Histopath samples – proper and
slip in 2 copies tightly closed container (depends on
4. Payment (cashier) size); slides with label
5. Sample collection/receiving and labeling ➢ Indicate also the date and time
6. Sample to main laboratory received in the charge slips

Phlebotomy Releasing of results or TAT

1. Patient Identification -always ask the patient 1. Routine test (CBC, UA, FA) – 1 hr
2. Double check the laboratory test to be tested 2. Chemistry with elec – 2 hr
3. Sample collection (syringe method) 3. Chemistry with hba1c – 11 am
4. Sample dispensing (order of draw) 4. Serology – 1.5-2 hr
5. Labeling with initials of the phlebotomist 5. TSH, FT4, FT3 – 2-3 hr
Syringe method 6. TPSA – 3 hr
7. Send out – the next day (Monday to Friday); if
Order of draw: Saturday – Monday after lunch
1. Yellow 8. Bacteriology – 5 days (for blood – 7 days)
2. Blue 9. Histopath – 7 working days
3. Red/Yellow
4. Green
5. Purple Causes of Hemolysis (hemolyzed specimen):
6. Gray/Black 1. Using a needle that is too small
Note: Dispensing of blood – Cap open and dispense 2. Pulling a syringe plunger back too fast
appropriate amount of blood
3. Expelling the bloed strongly into a tube

4. Forcing the blood from a syringe into an evacuated

Criteria for rejection tube
1. Insufficient sample 5. Shaking or mixing the tubes vigorously
2. Hemolyzed sample
3. Not in proper container 6. Performing blood collection before the alcohol has
4. Clotted sample dried at the collection site
5. Contaminated sample

Causes of Hematoma:

1. The vein is fragile or too small for the needle size.

2. The needle penetrates all the way through the vein.

3. The needle is partly inserted into the vein.

4. The needle is removed while the tourniquet is still

on.

5. Excessive probing.

6. Pressure is not adequately applied after

venipuncture.

III. SKIN PUNCTURE

➢ A fingerstick to obtain blood for routine

laboratory analysis is usually preferred for
children clder than one year old.
➢ Length of the lancet: 1.75mm (preferred; to
avoid penetrating the bone)
➢ The depth of the incision should be < 2.0 mm
for infants and children, and < 2.5 mm for
adults, to avoid contact with the bone.
➢ The distance from the skin surface to bone or
cartilage in the middle finger is 1.5-2.4mm.
The cut should be oriented across the
fingerprints to generate a large drop of blood
using a single deliberate motion.

Preferred Sites:

1. Lateral plantar heel surface-newborn

2. Palmar surfaces of the fingers (3 and 4th fingers)

3. Plantar surface of the big toe

4. Earlobes-least site
 CLINICAL CHEMISTRY Vitros 250 – dry-slide technology

Machines: - The analyzer is capable of accurately and

EL-120 – Electrolytes (Na, K, Cl, ionized Ca) quantitatively measuring 36 analytes in a
single serum, plasma, urine, or other
Vitros 250 – Blood chemistry (Chem 10, ALP, Ca, TP,
biological fluid with the capacity to run 40
Alb, LDH), Body fluids (Pleural, Peritoneal, CSF,
samples simultaneously. It can generate 250
Synovial)
tests per hour.
- Stand control for 15 minutes before using it
Main parts:

- Track
For BODY FLUIDS (pleural, peritoneal, CSF, synovial etc.) - Proboscis/Sample aspirator
- Incubator
Transfer sample in a clean plain test tube from the
- Processing unit and monitor
Bottle/Tube #1, centrifuge the specimen for 10minutes
and process, same as serum, in the machine using the Principle:
supernatant of the sample. Result is manually encoded,
- Reflectance Spectrophotometry - Light is reflected
and White Paper is used.
from the surface of a colorimetric reaction then used
to measure the amount or concentration of the
analyte
Rerun sample;
- The tests types are colorimetric, enzymatic end
1. if POTASSIUM is >6.0, delta check and verify sample. If point, two-point or multi-point rate, or potentiometric
no previous result, double check and rerun the sample.
- The amount of the reflected light depends on the
2. If SODIUM is >150 and <120, delta check and verify amount of chromophore formed but is not directly
sample. If no previous result, double check and rerun the related to the concentration of the analyte in the
sample. sample

Other analyzers:
Rerun sample; • POCT
1. if Creatinine is >3, delta check, verify diagnosis. If no • Urinalysis: using reagent strips
previous, double check and rerun the sample (dilute, if
needed only).

2 if ALT/SGPT is very high (>300), and AST/SGOT is within Electrolytes - Electrolytes are minerals that carry an
the normal range, double check and rerun the sample. electric charge.

3. if AST/SGOT is very high (>300), and ALT/SGPT is within - Electroneutrality: balance of charges wherein there
the normal range, double check and rerun the sample. is always an equal number of cations and anions

4. BILIRUBIN Sodium

a. For newborn (<14days), select BuBe only. - Aka natrium

b. If Be is 0, add 0.1 - Major cation of extracellular fluid

c. For Adult (>14days), select TBil, BuBc. - Contributes largest effect on Osmotic
Pressure.
d. If Be is 0, TBIL - Bu.
Potassium

- Aka kalium

- Most important analyte in terms of

abnormality being immediately life threatening
 - Major intracellular fluid cation involve or
very critical in electrical conduction of nerve impulses
or cardiac electrical conduction.

- any level of hemolysis will falsely increase

serum potassium result.

Chloride

- Major ECF anion

- The chief counter ion of sodium in CEF

- Chloride levels change proportionally to

sodium

- The only anion to serve as an enzyme

activator

Calcium

- Almost exclusively present in the plasma

- Involved in blood coagulation, excitability of

skeletal and cardiac muscle

- three forms: 50% free (ionized), 40% bound

to protein, 10% bound to anions

Erma EL 120

Principle: Ion selective electrode (ISE)

Sample Volume ≤ 90µL

Analysis Time ≤ 60 secs

Throughput ≤ 60 samples per hour

Precision

- CV %
- Na+ : ≤ 2%
- K+ : ≤ 2%
- CI- : ≤ 2%
- iCa++: ≤ 3%
- pH : ≤ 2%

Quality Control Tri-Level Quality Control, with L J Plots

of last 30 days
SEROLOGY 3 drops buffer

MINI VIDAS TSH, FT4, FT3, TPSA, FERRITIN, D-DIMER

- Principle: Enzyme Linked Fluorescent Assay H. PYLORI

(ELFA)
1 drop serum
GETEIN - SOB PANEL, PCT, CRP
1 drop buffer
- Principle: Quantitative immunoassay
15 minutes
KITS - HBsAg, HBV Ab, HCV Ab, RPR/VDRL, DENGUE
DUO, TYPHIDOT, H. pylori
HBsAg

100uL serum for 20 minutes

GETEIN

SOB PANEL
HIV 1 AND 2
1. 3 in 1 - 120uL of serum/citrated plasma
2. NT-proBNP - 120uL of serum/citrated plasma 10uL serum
3. D-dimer - 120uL of citrated plasma only
4 drops buffer
TROPONIN I - 120uL of serum sample only
15-20minutes
PROCALCITONIN - 120uL of serum sample only

C-REACTIVE PROTEIN
HAV IGG/IGM
1. 10uL serum to diluent
2. 120uL mixture to Cartridge 5uL of serum

Specimen stability for SOB PANEL and TROPONIN 1-4 2 drops buffer
HOURS only 15 minutes

MINI VIDAS HCV AB

TSH-200 uL of serum 10uL of serum
FT4-100 uL of serum 4 drops buffer
FT3-100 uL of serum (Conversion factor: 0.651) 20 minutes
TPSA-200 uL of serum

Human Ferritin 100 uL of serum Syphilis

D-dimer - 200 uL of citrated plasma 10ul plasma/serum / 20ul whole blood

4 drops buffer
No hemolyzed sample 15-20 minutes

DENGUE DUO Typhidot

Ns1: 2 drops serum 1 drop sample
1 drop buffer 1 drop buffer
IgG/IgM: 0.5uL serum 15 minutes
CLINICAL MICROSCOPY FECAL IMMUNOCHROMATOGRAPHY TEST (FIT)

MANUAL URINALYSIS ➢ Stick from 5 different sites and mix it with the
buffer.
➢ Check for the label and the charge slip
➢ Add 2 drops of the mixture to the kit
➢ Check for the sample volume if sufficient
➢ Read after 10 minutes
➢ Check for the Color and Transparency; record
➢ Report
➢ Do the dipstick; record
➢ Centrifuge sample (atleast 12mL) for 5
minutes; decant and smear in the slide
FECAL OCCULT BLOOD TEST (FOBT)
➢ Read and record
➢ SMEAR specimen (from 5 different sites)
thinly.
UF 500i ➢ Add 2 drops of buffer in the test area.
➢ Add 1 drop in the positive control
- Principle: fluorescence flow cytometry with
➢ Add 1 drop in the positive control
diode laser and hydrodynamic focusing
➢ Read after 3 minutes; Report
conductometry
- Throughput: normal mode: up to 60
samples/hour; special (high precision
bacteria) mode: up to 45 samples/hour

NOTE: execute SHUTDOWN for atleast 1 hour Stand

control for 15 minutes before using it

BODY FLUID CELL COUNT/DIFFERENTIAL COUNT

Cell Count

1. Uncentrifuged specimen charge into the

Neubauer Chamber.
2. Read WBC - 4 CORNERS
3. Read RBC - 5 CENTER SQUARES

Differential count

• Centrifuged specimen for 10 minutes

• Smear sediment into the slide and dry
• Stain using HemaQuick Stain; Dry
• Read

FECALYSIS

➢ Check for the label and the charge slip

➢ Check the Color and Consistency; record
➢ Smear (from 5 different sites), emulsify and
then read
➢ Report
HEMATOLOGY 4. HGB: Cyanide-free SLS method

CRITERIA FOR CONFIRMING RESULT BY MANUAL QC Materials

METHOD
Calibrator XN Cal
Hemoglobin: adult - 8 and below; 17 and above;
Control XN-L Check
newborn 10 and below
XN Check BF
WBC count-3 and below; 25 and above

Platelet count: 100 and below; 700 and above

MISPA i3 – single
note: Double check previous result, diagnosis and
correlate to other results - Principle: Rate based Nephelometry &
Immuno-turbidimetry methods
- 23 parameter assay model.
XN 550 & XN 350 – stand control for 5 minutes before 1. Stand reagent for 10-15 mins at room temp
using it before processing
2. 10 uL sample into diluent
3. 2 minutes
XN 550 4. 80 uL to cartridge
5. process
Throughput - up to 60 samples/h in WB mode; up to
70 samples/h in WB mode with the optional Speed-up D-10 – batch
licence
- utilizes gold standard ion-exchange HPLC
Measurement principles technology
1. Control machine before running (5uL control
1. WBC DIFF/RET: Fluorescence Flow Cytometry plus 1500uL diluent)
2. WBC: Flow Cytometry 2. Run sample into the machine (up to 10 spx)
3. RBC/PLT: DC Impedance method with 3. If microcontainer, 1500uL diluent/wash
hydrodynamic focussing solution plus 10 uL sample using cuvette.
4. HGB: Cyanide-free SLS method 4. Mix gently before running

NOTE:
XN 350 For specimen received from 6AM to 8AM and IN-
Whole Blood PATIENT, run sample in MISPA i3- RESULT after 2hours

- WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, For specimen received from 8AM to 10AM, BATCH
RDW-SD, RDW-CV, PDW, MPV, P-LCR, PCT, running using D10 - result is 11AM
NEUT#, LYMPH#, MONO#, EO#, BASO#, *sample stability is 12hours (not hemolyzed)
NEUT%, LYMPH%, MONO%, EO%, BASO%, IG#,
IG%

With RET (optional) CA-104

- RET#, RET%, IRF, LFR, MFR, HFR, RET-He, PLT-O - Principle: Clotting: turbodensitometric photo-
optical measuring principle
Body Fluids with XN-BF (optional) ➢ PT: Dade Innovin, Thromborel S
- WBC-BF, RBC-BF, MN#, PMN#, MN%, PMN%, ➢ APTT: Dade Actin, Dade Actin FS, Dade Actin
TC-BF# FSL, Pathromtin SL
➢ Fibrinogen: Multifibren U
Measurement principles ➢ Thrombin Time: Test Thrombin,
1. WBC DIFF/RET: Fluorescence Flow Cytometry Thromboclotin
2. WBC: Flow Cytometry ➢ Factor Deficiency: Factor II, V, VII, VIII, IX, X, XI,
3. RBC/PLT: DC Impedance method with XII
hydrodynamic focussing
Prothrombin Time WBC 45 – 10.5

1. Set PT mode SEG 30.0 – 70.0

2. Incubate cuvettes for pre-warming (10 mins)
LYMP 20.0 – 40.0
3. Add 50 ul of Patient’s plasma
4. Place in measuring channel MONO 0.00 – 12.0
5. At “GO-S” dispense 100ul of PT reagent
(thromborel S or Dade Innovin) EOS 0.00 – 6.0

Activated Partial Thromboplastin Time BASO 0.0 – 5.0

1. Set APTT mode PLT 150.0 – 450.0

2. Incubate cuvettes for pre-warming (10 RDW-SD 37.0 – 54.0
minutes)
3. Add 50ul of patient’s plasma + 50ul APTT RDW-CV 11.0 – 16.0
reagent (Pathromtin SL or Actin FSL)
4. Place in measuring channel
5. At “GO-S” dispense 50ul of Calcium Chloride Neutrophil Bacterial/Fungal

Note: Pre-warm cuvettes (10 minutes) before using. Eosinophil Parasitic

Stand reagent 15 mins before placing in the incubator
Basophil Allergic Response

Monocyte Phagocytosis
Control and Calibrators (CA-104)
Lymphocyte Viral
1. Citrol (1E and 2E)
- Reconstitute with 1mL distilled water Stand
for 15 mins prior to use Falsely High RBC Falsely Low RBC
- Opened Stability: 16 hours 1. Large platelets 1. Cold agglutinins
2. Standard Human Plasma interference 2. EDTA-dependent
- Reconstitute with 1mL distilled water 2. Marked agglutination
- Stand for 15 mins prior to use thrombocytosis 3. RBC lysis due to
- Opened Stability: 4 hours 3. Fragmented red mishandling
cells 4. extreme
3. Owren's Veronal Buffer (Diluent)
microcytosis
- Opened Stability: 2 months

Falsely High Platelet Falsely Low Platelet

Erythrocyte sedimentation rate (ESR) 1. Microcytic RBC 1. partial clotting of
2. Fragmented red cell blood sample
- Westergren tube 2. activation of PLT
- 3mL sample using Sodium citrate tube. Use during venipuncture
ESR tube, read after 60mins 3. EDTA-induced PLT
aggregate
4. PLT satellitism
Reference value 5. giant platelets

RBC 4.0 – 6.0

HGB 12.0 – 15.0

HCT 37.0 – 47.0

MCV 86.0 – 110.0

MCH 26.0 – 38.0

MCHC 31.0 – 37.0

BLOOD STATION

1. ABO/Rh Typing Reverse

2. Crossmatching
1. Centrifuge (serum/yellow top)
3. Coomb’s test
2. 2 drops of serum in a tube
4. Red cell suspension preparation (A cells and B
3. 2 drops of known A and B
cells)

BLOOD TYPING (TUBE METHOD) COOMB'S TEST (GEL METHOD)

1. Label test tubes properly as follows; INDIRECT
a. A 1. 50 uL of O Rh positive Red Cell Suspension
b. B
c. D 2. 25 uL of patient's plasma
d. Ac 3. Incubate for 15 minutes *
e. Be
4. Centrifuge
2. Make 3-5% red cell suspension by adding

a. 1-2 drops of washed red cells

b. 19 drops buffered NSS or NSS DIRECT

3. Place reagents on the appropriate tubes; 1. 50 uL of patient's Red cell suspension

a. 1 drop of Anti-A to tube "-A" 2. Centrifuge

b. 1 drop of Anti-B to tube "-B"
c. 1 drop of Anti-D to tube "D"
d. 1 drop of Al cells to tube Ac CROSSMATCHING (TUBE METHOD)
e. 1 drop of B cells to tube Be
1. Label test tube properly (Patient's name, Segment
4. Add 1 drop of 3-5% red cell suspension to the first 3 No.)
tubes
2. Add 2 drops of patient's serum/plasma
5. Add 2 drops of serum or plasma to tubes Ac and Be
3. Add 1 drop of 3-5% donor's red cell
6. Mix gently and centrifuge for 30 seconds
4. Centrifuge for 30 seconds
7. Gently re-suspend the red cell button and examine
5. Read macroscopically and record the result -
for the agglutination
Immediate Spin
8. Read and record the result and reactions
6. Add 2 drops of LISS

7. Mix and incubate for 10-15 minutes at 37°C

NOTE: Proper reporting of Blood type is in complete
8. After incubation, centrifuge for 30 sec immediately
words e.g: O Rh positive OR O positive
9. Read macroscopically and record the result -
Thermo phase (37°C)
Forward
10. Wash 3-4x with large amount of buffered saline
1. Put NSS in a separate tube (3/4 full)
2. Drop 2 (A,B,D) separate tube
11. After the last washing, blot the tube in a gauze
3. Then 2 drops of whole blood. Mix
pad/tissue paper to absorb excess saline
4. Then 2 drops of RCS (NSS+2 drops of EDTA
blood) 12. Add 2 drops AHG reagent
5. Centrifuge for 1 min
13. Spin for 30 sec and read immediately
6. Read result
microscopically - AHG phase
14. Record results and interpretation and grading of
reactions
A CELLS AND B CELLS PREPARATION

1. Prepare known blood type for the suspension

OR
2. Label tubes with A, cells and B cells and Date

3. Wash each suspension

1. Set the unit in the refrigerator
* Wash 3-4x with large amount of buffered saline (3/4
2. Write serial number/CD/ED/3 digits of
full)
segment, blood type in charge slip
3. Label tubes (Px RBC EDTA, Donor 3 digits of 4.Store
segment)
4. Centrifuge EDTA 5 mins
5. Cut segment from Blood bag put in the donor LISS - low-ionic-strength saline
tube
6. Put diluent (1000mL/1 punch) - used in blood bank testing to potentiate
7. 10ul -> blood from segment to donor tube reactions between antibodies and red blood
8. 10 ul plasma -> to px tube cells
9. 50ul -> patient RCS to microtube 1,2,3,6 - reates a net positive electrical charge, and the
10. 50ul -> donor RCS to microtube 1,2,3,4,5 (A, B, similar charge on all RBCs causes them to
D, Control, AHG) repel each other (this is also known as “zeta
11. 25ul -> Patient plasma to microtube 5,6 potential”)
12. Incubate for 15 minutes - contains fewer sodium ions than “normal”
13. Centrifuge card for 10mins saline, fewer Na+ ions are available to
14. Read and interpret result congregate at the RBC surface, and thus, the
zeta potential is lessened, allowing antibodies
to work more efficiently to agglutinate
Manual Crossmatching procedure incompatible red cells.

1. Add 2 drops of patient’s plasma/serum with 1

drop of 3-5% donor’s RCS Interpretation of Agglutination Reactions
2. Centrifuge 30 seconds Macroscopically Designation Background
3. Read (immediate spin) observe findings
4. Add 2 drops of LISS One solid 4+ Clear
5. Mix and incubate at 37oC for 15 mins agglutinate
6. Centrifuge for 30 seconds Several large 3+ Clear
7. Read (thermos phase) agglutinate
8. Wash 3-4 times with NSS Medium-sized 2+ Clear
9. After the last washing, blot in gauze or tissue agglutinate
10. Add 2 drops of AHG Small 1+ Turbid
agglutination
11. Spin for 30 seconds
No agglutination 0 Turbid
12. Read (AHG)

Incubator – 37oC, 15 mins

3-5% RED CELL SUSPENSION PREPARATION
Centrifuge – 10 mins
1. Wash red cells 3 - 4x.

***Wash 3-4x with large amount of buffered saline

(3/4 full)

2. Add 1-2 drops of

3. 19 drops of N
 Respiratory specimen

MICROBIOLOGY EC LPF <25

1. Culture and sensitivity PC LPF >25

2. Gram stain
Organism OIO 1+, 2+, 3+, 4+
3. Acid fast bacilli (AFB)
4. KOH LPF: Bartlett’s Method

Bacteria = grading

Yeast = mod, few

Sputum – bartlett’s

WBC >25 pus cells

EC < 25 cells

Bacteria: Grading

1+, 2+, 3+, 4+

Yeast Rare, few, moderate, many

Urine

WBC Range (ex. 0-2)

EC Rare, few, moderate, many

Yeast Rare, few, moderate, many

Pleural Fluid
AFB Collection WBC and organism
- First thing in the morning deep couch (no
water/food). Then after 1 hour collect again
Others:

EC Rare, Few, Mod, Many

AFB Reading Scale
0 No AFB seen in 300 OIF PC LPF >25
+n 1-9 AFB/100 OIF Organism OIO 1+, 2+, 3+, 4+
1+ 10-99 AFB / 100 OIF
2+ 1-10 AFB/OIO in at least 50 fields
3+ More than 10 AFB/OIO in atleast 20 fields
Blood Bottles

5 below 1mL

Adults 5mL – min

10mL – max
Blood c/s – 24 hours, 3rd day, 5th day Gram (-) 0.50 – 0.63

Urine – 48 hours, final Gram (+) 0.50 – 0.63

ETA/Sputum, Wound/Stool, Transudates – 3 days Yeast 1.8 – 2.2

CSF – 5 days NH/ ANC 2.7 – 3.3

Specimen 37oC Candle Jar RT MAC

Blood Blood
culture 17.5g
bottle
Replant to: MAC BAP, CAP
Urine Uris or
BAP,
MAC
Stool/Rectal MAC, APW/
swab SSA. Selenite
TCBS
CSF Blood
culture
bottle + -
Replant to: MAC BAP/CAP Aeromonas hydrophila, Actinobacillus spp.,
Throat BAP/CAP Aeromonas punctata, Aeromonas salmonicida,
Swab Bacillus alvei, Alcaligenes sp., most
ETA/Sputum MAC BAP/GBA/BCA Edwardsiella sp., Bacillus sp., Bordetella
*Add Escherichia coli, sp., Enterobacter sp.,
optochin to Flavobacterium sp., most Haemophilus sp.,
GBA Haemophilus influenzae, most Klebsiella sp.,
Wound/ THIO or BACP, CAP Klebsiella oxytoca, Neisseria sp.,
Eye/Ear TSB, Proteus sp Mannheimia
Discharge MAC haemolytica, Pasteurella
Urethral MAC CAP, MTM, ureae, Proteus mirabilis
Discharge BAP
Transudates Blood
(peritoneal, culture
pleural, bottle
synovial)

Replant to: MAC BAP, CAP

Vaginal MTM, CAP
Discharge

Gram stain

Crystal violet – Primary

+ -
Gram’s iodine – mordant Klebsiella, Enterobacter , Escherichia coli
Ethyl alcohol/acetone – decolorizer Citrobacter, Providencia, Yersinia enterolitica
Proteus, Serratia, vibrio Salmonella typhi
Safranin O – counterstain cholerae, Pseudomonas, Edwardsiella tarda
Salmonella enteritidis Vibrio hollisae
and members of the
Germ tube subgenera Salmonella II,
III and IV.
Red top + citrated plasma
Germ tube (+) – C albicans (lollipop)

Germ tube (-) – C. tropicalis, C. glabrata

α β γ
S. pneumoniae S. aureus E. fecalis
S. viridans S. pyogenes S. saprophyticus
- oxidase positive bacteria contain cytochrome S. agalactiae S. epidermidis
c oxidase and produce a change in color of the
reagent from colorless to bluish or purplish in
less than 30 seconds

+ -
Pseudomonas Enterobacteriaceae
Neisseria Acinetobacter
Vibrio Staphylococci
Campylobacter Streptococci
Aeromonas
Brucella
Pasteurella
Eikenella
Kingella
Moraxella
Legionella Lactose Fermenter Non-lactose Fermenter
Helicobacter E. coli Edwardsiella tarda
Enterobacteriaceae Proteus mirabilis
Klebsiella Proteus vulgaris
Salmonella enterica
Shigella spp. (boydii,
dysenteriae, flexneri)
Shigella sonnei
Yersinia enterolitica
Yersinia pestis

+ -
Nocardia Streptoccoci
Pseudomonas Micrococcus
Listeria
Aspergillus
Candida
E. coli
Staphylococcus
Serratia
B. cepacia
 Gas production:demonstrated by the presence of
bubbles or cracks in the medium

- Lysine iron agar (LIA) slants tests organisms for

the ability to deaminate lysine or
decarboxylate lysine. Lysine deamination is an
aerobic process which occurs on the slant of
the media. Lysine decarboxylation is an
anaerobic process which occurs in the butt of
the media.
- Lysine iron agar. A, Alkaline slant/alkaline butt
(K/K). B, Alkaline slant/alkaline butt, H2S
positive (K/K H2S+). C, Alkaline slant/acid butt
(K/A). D, Red slant/acid butt (R/A). E,
Uninoculated tube.

Lysine Decarboxylation (detected in butt):

Positive Test: Purple slant/purple butt

(alkaline), the butt reaction may be masked by H2S
production

Negative Test:Purple slant/yellow butt (acid),

- A positive test for indole is indicated by the
fermentation of glucose only
formation of a pink to red color band at the
top of the medium after the addition of
Kovacs' Reagent. If a yellow color remains, this
Lysine Deamination (detected on slant): indicates a negative indole test.
Positive Test:Red slant

Negative Test:Slant remains purple

H2S Production:

Positive Test:Black precipitate

Negative Test:No black color development

 - The urease test identifies those organisms
that are capable of hydrolyzing urea to
produce ammonia and carbon dioxide

+ -
Proteus Escherichia coli
Nocardia
Ureaplasma
H. pylori
- Triple Sugar Iron Agar is used for the Klebsiella
presumptive identification of Cryptococcus
Enterobacteriaceae based on the S. epidermidis
fermentation of glucose, lactose, sucrose and S. saprophyticus
the production of gas and H2S.
- Triple Sugar Iron Agar contains three
carbohydrates (glucose, lactose and sucrose)

Principle:

1. When one of the carbohydrates is fermented,

the lowering of the pH will cause the medium
to change from reddish orange (the original
color) to yellow. A dark red color indicates
alkalinization of the peptones.
2. Sodium thiosulfate in the medium is reduced
by some bacteria to hydrogen sulfide (H 2S),
the latter reacts with ferric ions in the
medium to produce iron sulfide, a black
insoluble precipitate.
 2. Xylene – 3 mins
3. Absolute alcohol – 2 mins
HISTOPATHOLOGIC TECHNIQUES AND CYTOLOGY
4. Absolute alcohol – 2 mins
1. GROSSING 5. 95% ethyl alcohol – 2 mins
2. FIXATION 6. Tap water – 5 dips
3. DEHYDRATION 7. Harris hematoxylin – 20 mins
4. CLEARING 8. Tap water – 5 dips
5. EMBEDDING 9. 1% acid alcohol – 5 dips
6. CUTTING 10. Tap water – 5 dips
7. MOUNTING 11. Ammonia water – 3 dips
8. STAINING 12. Tap water – 5 dips
13. 80% alcohol – 5 dips
14. 95% alcohol – 5 dips
Oven – 65oC 15. Eosin – 10 dips
16. Absolute alcohol – 1 min
Tissue embedding – 55, 60, 65, 70oC 17. Absolute alcohol – 1 min
Celltrazone – for LBC and non-gyne 18. Xylene – 1 min
19. Xylene – 1 min
Citadel 2000 – Tissue processor 20. Air dry – 5 min
- Specimens are automatically raised from their
reagent containers every 10 minutes and then
reimmersed Tissue Processing

1. 10% Neutral buffered formalin

2. 10% Neutral buffered formalin
Papanicolaou staining (papsmear & cytology) 3. 70% ethyl alcohol
4. 80% ethyl alcohol
1. 95% ethyl alcohol – 10min
5. 95% ethyl alcohol
2. Running tap water – 5 dips
6. Absolute alcohol
3. Harris’ hematoxylin – 10min
7. Absolute alcohol
4. Tap water – 5 dips
8. Xylene
5. OG 6 – 3 min
9. Xylene
6. 95% ethyl alcohol – 5 dips
10. Xylene
7. EA 50 – 3 mins
11. Paraffin (1)
8. 95% ethyl alcohol – 5 dips
12. Paraffin (2)
9. 95% ethyl alcohol – 5 dips
13. Remove from wax
10. Tap water – 5 dips
11. Air dry

Thickness: 4-6 (gregorio’s)

Giemsa Staining Clearance angle – 2-8 degrees
1. Methanol – 3mins Coverslip:
2. Tap water
3. Eosin – 2mins 1. 24x60 – cytology
4. Tap water 2. 24x50 – surgical
5. Methylene blue – 2mins Harris – mercuric oxide
6. Tap water
7. Air dry Meyer’s – progressive staining/no acid alcohol after

Hematoxylin – nuclear

H&E staining – regressive Eosin – cytoplasmic

1. Xylene – 3 mins Water batch temp – 45-50oC

Cytology fixative – 95% alcohol