CLINICAL INTERNSHIP – CLINICAL MICROBIOLOGY

DAY 2 – GRAM STAIN & AFB

By: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the Golden-Winged King

GRAM STAINING Serotype (group A vs B vs D strep)

- Is the most important staining procedure in
o Biologically inert – Shigella
microbiology
o S. pyogenes – Schultz-Charlton test
- It is a differential staining procedure used to differentiate
o Salmonella (Enteric fever)- Widal
two large groups of bacteria based on their different cell
o Rickettsia - Weil felix test
wall constituents
 OX-K come from Proteus mirabilis
o Mycoplasma does not have cell wall and cannot be
 OX-2 & OX-19 come from Proteus vulgaris
gram stained
o Tuberculin test and Mantoux test for Mycobacterium
o Mycobacteria has mycolic acid which does not
o Dick’s Test → Scarlet fever
respond to gram stain
o Mallein test → Glanders disease
o Legionella requires sliver stain
o Schick test → Diphtheria
o Chlamydia (intracellular parasite)
o Ascoli test → Anthrax
 smallest bacteria
- Antibiotic resistance patterns (MSSA vs MRSA)
o Rickettsia (intracellular bacteria)
o Mueller-Hinton Agar, Kirby Bauer
- This procedure used to distinguish between Gram
- rRNA sequence analysis
Positive organisms and Gram Negative organisms
BACTERIAL MORPHOLOGY
CLASSIFICATION OF BACTERIA
- Along with gram staining, general morphology are the
- Morphology (Cocci vs Rod/Bacilli vs Coccobacilli)
most important methods of differentiating bacteria
- Gram stain (Positive vs Negative vs Variable)
o Reporting example: Presence of Gram positive cocci
in cluster, Presence of gram positive cocci in chain
and in pairs; whoever is most prominent is usually
the one reported
- Growth requirements (aerobic vs anaerobic)
o Oxygen
o Temp requirements: Mesophilic (most pathogenic
bacteria); same with body temp
o Doubling time
o Nutritional requirements: Agar
- Biochemical reactions (lactose fermenting vs non
fermenting)
o For identification of Gram negative organisms
o ONPG, Mac-Conkey Agar, Catalase test, Hemolysis
pattern in blood agar, etc.
 - Gram positive bacilli
o Clostridium, Actinomyces, Mycobacterium,
Nocardia, Corynebacterium, Lactobacillus,
Erysipelothrix, Bacillus
- Gram negative bacilli
o E. coli, Proteus, Klebsiella, Serratia, Enterobacter,
Shigella, Salmonella, Yersinia, Pseudomonas,
Stenotrophomonas, Vibrio, Bacteroides
- Gram negative cocci
o Neisseria, Moraxella, Veilonella
- There are no bacilli in chains - Gram (+) coccobacilli
o Listeria
CELL WALL OF BACTERIA - Gram (-) coccobacilli
o Haemophilus
o Legionella
o Bordetella
- Gram variable
o Acinetobacter

- Gram positive: Thick peptidoglycan, lack lipoproteins

- Gram negative: Thin peptidoglycan, lipoproteins
- Gram positive cocci:
o Staph: S. aureus, S. pyogenes, S. saprophyticus, S.
epidermidis
o Strep: S. pyogenes, S. pneumoniae, S. viridans, S.
agalactiae
o Enterococcus species
o Peptostreptococcus
EQUIPMENT NEEDED FOR GRAM STAINING o Lastly, before recapping the test tube, allow the lip
of the test tube to pass through again into a Bunsen
- Bunsen burner
burner to keep from contamination
- Coplin Jar/Staining Rack
- Solid media – plate
- Inoculating loop
- Liquid Media (broth) – Tube
- Microscope
- Stain/Slide rack 3. AIRDRY AND FIXATION: Air dry then heat fix the sample
- Alcohol-cleaned microscope slide (frosted/non-frosted) to the slide by carefully passing the slide through a
o Frosted: Ideal Bunsen burner 3 times
- Pencil

REAGENTS NEEDED FOR GRAM STAINING

Reagent Function
Primary stain: Stains all bacteria blue to purple
Crystal Violet
Mordant: Enhances reaction between cell wall and
Iodine solution/ primary stain
Gram’s iodine
Decolorizer: Gram (+): Retain the primary stain
4. PRIMARY STAIN: Add crystal violet to the sample/slide
Ethyl alcohol because of the peptidoglycan and
and settle for one minute. Rinse the slide with a gentle
(Ethanol) or teichoic acid cross-links
stream of water for a max of 5 seconds to remove
Acetone Gram (-): lose the primary stain because
unbound crystal violet
of the large amount of lipopolysaccharide
in the cell wall 5. MORDANT: Add Gram’s iodine then settle for one
Counterstain: No effect on gram-positive bacteria; minute. This will serve as mordant or an agent that fixes
Safranin O or stains gram negative bacteria pink to red the crystal violet to the bacterial cell wall
carbolfuchsin
Water Preferably in squirt bottle
Distilled water is used, not tap water 6. DECOLORIZATION: Rinse sample/slide with Acetone or
Alcohol for 3 seconds and rinse with a gentle stream of
PROCEDURE water.

1. LABELLING: Label the frosted slide with patient’s name 7. COUNTERSTAIN: Add safranin (secondary stain) to the
using pencil. Smear (circular motion) the bacteria using slide then settle for 20-30 seconds (book: 60 seconds).
inoculating loop on glass slide to be stained. Wash with gentle stream of water for 5 seconds. Then,
o Add NSS, then place the inoculum let the sample air dry for microscopic examination
o No NSS is added in hospital setting
o The slide must be passed through the Bunsen burner
for 2-3 times before placing the inoculum

2. STERILIZATION AND TRANSFERRING: For solid media,

before taking an isolate colony (3rd or 4th quadrant) from
agar, flame your loop for sterilization then allow your
loop to cool. Then, transfer a small amount of growth
from the medium to the slide and emulsified using a
drop of sterile water or NSS (saline)
o To determine if the nichrome wire is completely
sterile, the fire must turn red
o Allow the loop to cool

- Liquid Media
o Before taking a colony from the broth, flame your - Coverslip prevents contact between the objectives and
loop for sterilization then allow your loop to cool. To the specimen
prevent from contamination, after opening the test - Examine in the microscope under an OIO
tube, allow the lip of the tube to pass through a - When using OIO, don’t use coarse adjustment knob, only
Bunsen burner 2x to 3x then you may get a loopful use the fine adjustment knob
of sample. - Low power and scanner, open the diaphragm
 GRAM STAINING PROECURE FROM DELOST:

PURPOSE

- To categorize
bacteria as gram positive or
gram negative and to observe
the cellular morphology. Gram
stain is also a valuable tool in
the direct observation of
clinical material.

- Presence of gram positive cocci in clusters

PRINCIPLE
- Bacteria will differentially retain the primary stain based on the
characteristics of the cell wall. Gram-positive bacteria, with a
high content of peptidoglycan and teichoic acid, retain the
primary stain and appear blue to purple. Gram-negative
bacteria, with a high content of lipopolysaccharide in the cell
wall, lose the primary stain during decolorization. Gram-
negative bacteria take up the counterstain and appear pink to
red.
- Presence of gram negative bacilli
MATERIALS
- Crystal violet
- Gram's iodine
- Acetone or ethyl alcohol
- Safranin O
- 95% methanol

PROCEDURE
1. Prepare a thin smear of the culture or specimen to be observed.
Allow the smear to air dry and heat fix. Alternatively, fix the
smear by flooding with 95% methanol and allowing to air dry.
2. Overlay smear with crystal violet for 30 seconds to 1 minute.
3. Rinse with distilled water, tapping off excess.
4. Flood smear with Gram's iodine for 1 minute.
- Presence of gram positive diplococci, or cocci in pairs 5. Rinse with distilled water, tapping off excess.
6. Add acetone or ethyl alcohol to decolorize drop by drop until
no violet color appears in rinse. This requires less than 10
seconds.
7. Rinse immediately with distilled water.
8. Flood smear with safranin O for 30 seconds.
9. Rinse with distilled water and allow slide to drain.
10. Blot dry with paper towel (bulbous paper) or air dry for delicate
smears.
11. Examine each slide under oil-immersion objective for
characteristic Gram stain reaction, morphology, white blood
cells, and other important structures.
- Presence of gram positive cocci in chains; most probably INTERPRETATION
Streptococci - Gram-positive organisms stain blue-purple.
- Gram-negative organisms stain pink-red.
REPORTING:
QUALITY CONTROL
- Gram positive cocci in clusters
- Gram positive cocci in chains and clusters - Prepare a mixed suspension of Staphylococcus (gram +) and
Escherichia coli (gram -) in saline. Place a drop on the surface
- Gram positive diplococci
of the slide and allow to air dry. Fix and stain smear as in above
- Gram negative coccobacilli
- method. Staphylococcus appear as purple cocci in clusters. E.
- Gram negative bacilli coli appear as pink bacilli.
AFB STAINING: DIRECT SPUTUM SMEAR MICROSCOPY - Bunsen burner/alcohol lamp
- Metal waste can
DIRECT SPUTUM SMEAR MICROSCOPY (DSSM) - Disinfectants
- Alcohol sand flask
- Is the most widely used means for diagnosing pulmonary
- Biosafety cabinet (optional); as long as the DOH has
tuberculosis and is available in most primary health-care
approved it
laboratories at health-center level
o TB can infect not just the lungs but also other organs DIRECT SMEAR PREPARATION
o Not inclusive to sputum but can also use other body
fluid 1. Numbering/labelling the slide
o Reporting: Qualitative (extra-pulmonary samples; no o Write down the yearly serial
grading) number or order number of
o Non-pulmonary and Extra-pulmonary tuberculosis sputum specimen on an
sample: no grading end of the slide (Year/
o Sputum requires grading Patient number/ Slide no.)
- It has been shown conclusively that good-quality o DOH doesn’t allow complete names due to
microscopy of two consecutive sputum specimens confidentiality
identifies the vast majority (95-98%) of smear-positive TB 2. Sputum smearing
patients o Fish out one loopful of yellowish particle of sputum.
- DOTS (Directly Observed Treatment, Short-course): make Spread one loopful of the sputum evenly on a clean
Ph TB free country labelled glass slide, approximately 2cm x 3cm in size
o Increase the number of laboratories that test for o It is important that the proper size is followed to
tuberculosis increase the detectability of acid fast bacilli
o Even primary level hospital/laboratory
o To increase the chance of detecting positive patient
- Widely used (Business permit, Job application, and
pregnant women)
o Pregnant women can’t undergo XRAY instead DSSM
is suggested
- Ideal number of sputum is 2 samples with an hour - Quality of sputum: Salivary, Mucosalivary,
interval Mucopurulent, Purulent
- Label a new clean, unscratched slide at one end with
PROCESS
the laboratory serial number
- Label frosted slides with pencil
- Label non-frosted slides with diamond pencil
- Use mucopurulent (thickness of the smear is easily
MICROSCOPIC
RECORDING AND achieved), opaque, grayish or yellowish portion for
SMEARING STAINING REPORTING OF
OBSERVATION
RESULTS smear preparation. Transfer an appropriate portion of
the specimen to the slide by using an applicator stick or
wire loop.

EQUIPMENTS, SUPPLIES, AND REAGENTS FOR SMEAR

PREPARATION
- Sputum container
- Coconut mid-rib/wire loop
o Coconut mid-rib tip is thin and coiling is performed
easier, the tip can also be cut; wire loop can be
reused but the tip is not thin
o Repeated coil type = even distribution,
o Sterile applicator sticks (single use)
even thickness of smear
o Wire loop - fast and efficient
o Saliva is prone to false negative result
- Glass slides
3. Removal of adherent sputum
o Glass slide - free from scratches and scuffs
o Dip the wire loop in a washing bottle and
o Don’t use reused slide
remove the excess sputum from the wire
- Marking pen
loop by moving it up and down in sand
o Pencil resists decolorization
- Forceps
- Match
Sterilization of the wire loop STAINING WITH ZIEHL-NEELSEN METHOD

o Heat the wire loop in a flame till - Ziehl-Neelsen – hot method, mordant is heat; Kinyoun –
red hot cold method
- Ziehl–Neelsen staining is a type of acid-fast stain, first
introduced by Paul Ehrlich Franz Ziehl (1859–1926) and
4. Drying and Fixation of the the pathologist Friedrich Neelsen (1854–1898).
Smear
o Allow the smear to dry 1. THE PRIMARY STAIN
completely at room o Pour carbolfuchsin stain to cover the whole surface
temperature and fix it by of the slide
passing through the
2. MORDANT
flame 2-3 times about 2-3 seconds each
o Heat the slide till steam comes off from the stain.
o Too much heating = bacteria will lose its acid
o Do not boil and do not allow the slide to dry.
fastness
o Leave it for 10 minutes
5. Arrangement of the slide o Alcohol lamp is better; when steam is released, stop
o Place the slide on the heating
staining bridge
o Remember to leave 3. REMOVAL OF EXCESS STAIN
enough space o Tilt the slide to drain off excess stain
between slides to
4. WASHING OF THE SLIDE
prevent transfer of
o Wash the staining solution off with a gentle stream
material from one
of running water
smear to another and
the solution running 5. DRAINING OFF EXCESS RINSE WATER
off from the slides o Tilt the slide to drain off excess rinse water
o Allow smears to air dry o Boiling – leads to false reading; only wait for it to
for 15 minutes. Do not steam
use heat for drying
o Pass slide through a flame three or four times with 6. DECOLORIZATION
the smear uppermost. Do not overheat. Allow to cool o Cover whole slide with acid alcohol or ethanol and
before staining leave it until no more color drains from the smear
o Discard the applicator stick in disinfectant and use a 7. WASHING OF THE SLIDE
new one for each specimen. Remove particles of o Wash the slide with gentle stream of running water
adherent sputum from wire loop by moving it up and
down through a sand alcohol bottle containing 70% 8. DRAINING OFF EXCESS RINSE WATER
alcohol. Flame wire loop thoroughly prior to re-use. o Tilt the slide to rinse excess water

EQUIPMENTS, SUPPLIES AND REAGENTS FOR ACID FAST 9. COUNTERSTAINING

STAINING o Pour methylene blue stain to cover the whole surface
of the slide and leave for 10-30 seconds
- Carbol fuchsin: Primary stain
o Stock alcoholic fuchsin: Basic fuchsin + 95% ethanol 10. REMOVAL OF METHYLENE BLUE
o 5% Phenol solution: Phenol solution + distilled water o Pour off methylene blue stain
o Ziehl’s solution: Stock alcoholic fuchsin + 5% phenol
11. WASHING THE SLIDE
- Acid alcohol/ethanol
o Wash the slide with a gentle stream of running water
o 95% ethanol or acid alcohol
 3% hydrochloric acid, 95% ethanol 12. DRYING THE STAINED SLIDE
- Methylene blue = counterstain o Tilt and place the slide on the slide rack to air dry
o Substitute: Malachite green o Thickness is enough pag clear pa ang news paper
- Staining bridge/rack o Note: the thickness is enough if the print of a
- Alcohol lamp newspaper is readable when the slide is
- Match superimposed on it
- Forceps
- Request forms
- Pencil/pen
- Paper towel
- Running water or wash bottle
 MICROSCOPIC EXAMINATION

- Application of immersion oil

- Observation of stained smear
o Examine the smear under 100x objective with 10x
eye piece lens
- Smear reading
o Read at least 300 visual fields to give a report as
negative. Record the result.

1. Scanner
AFB STAINING PROCEDURE ACCORDING TO DELOST 2. LPO
PRINCIPLE 3. Add Immersion oil
4. OIO
- The mycobacteria o Use fine adjustment knob only
do not decolorize
after staining, SMEAR READING
even with the
addition of acid or - Read the slide
acid-alcohol, systematically
because of the - Proceed from left to
mycolic acid in the right end to other end.
cell wall. Heat, Come down a little and
solvents, or then proceed either
detergents are
from left to right
needed to drive
- Current form of
the stain into the
cell wall of the reading: Horizontal
mycobacteria. - One horizontal line of a 3 cm width of the smear
corresponds to approximately 150 (100-150) visual fields
PURPOSE of magnification x 1000 (100x objective and 10x eye
- Mycobacteria, commonly known as acid-fast bacilli (AFB), are piece)
detected in clinical specimens and cultures through use of - Read two horizontal lines which correspond to 300 visual
carbolfuchsin staining. fields to report the slide as negative
- Read at least one horizontal line. When AFB is seen,
ZIEHL-NEELSEN METHOD MATERIALS
count the number of bacilli present. When AFB is not
- Carbolfuchsin stain: Basic carbolfuchsin in 95% ethanol in 5% observed on one horizontal line, continue to read on
phenol second horizontal line.
- Decolorizer: 95% ethanol in concentrated hydrochloric acid - Report as positive for acid-fast bacilli if present
- Counterstain: Methylene blue chloride in distilled water - Report as negative if no AFB seen for 300 visual fields
PROCEDURE

- This procedure must be performed wearing gloves, mask, and

gown in a class 2 biohazard hood.

1. Fix smears using an electric warmer at 65°C for 2 hours.

2. Place a small piece of filter paper over the smear.
3. Flood slide with carbolfuchsin and heat the electric warmer to
steaming for 15 minutes.
4. Remover filter paper and rinse slides with distilled water.
5. Decolorize with acid-alcohol until no more color drains from
the smear.
6. Rinse with distilled water.
7. Counterstain with methylene blue for 30 seconds.
8. Rinse with distilled water and allow to dry.
9. Examine using the oil-immersion objective (1OOx).

INTERPRETATION

- Acid-fast bacilli stain red, and the background should be blue.

NATIONAL STANDARD REPORTING SCALE

0 No AFB seen in 300 visual fields

+n 1-9 AFB/100 visual fields
1+ 10-99 AFB/100 visual fields
2+ 1-10 AFB/in at least 50 visual fields
3+ More than 10 AFB/in at least 20 visual fields
- +n to 3+ are positive readings
- If you counted 8 AFB per 150 visual fields, report as +8

MAIN CAUSES OF FALSE READINGS IN SMEAR

PREPARATION

CHECKPOINT CAUSES FALSE FALSE

POSITIVE NEGATIVE
Size Too big *
Too small *
Evenness Uneven *
Sloughed-off *
Thickness Too thick *
Too thin *
Cleanness Dirt * *
Artifact *
Sputum Saliva *
quality Blood stained *
- RIPE: Rifampin, isoniazid, pyrazinamide, and ethambutol Staining Overheating * *
Insufficient heating *
Under decolorization * *
Over decolorization *
- Take note: Size, Evenness, Thickness (DOH Focus)
- Ideal quality: Purulent and Mucopurulent

IMPACT OF FALSE-POSITIVE RESULTS

- Patients are started on treatment unnecessarily

- Tuberculosis medications are wasted
- In follow-up examinations, the intensive phase of
- Submitted quarterly to the department of health treatment is continued longer than necessary
- MDR: Multi-drug resistant; XDR: Extensively drug
resistant

IMPACT OF FALSE-NEGATIVE RESULTS

- Patients with tuberculosis are not treated, resulting in

suffering, spread of tuberculosis and death
- Intensive phase treatment is not extended for the
required duration, resulting in inadequate treatment

DOH TB DOTS TREATMENT

- Rifampicin
- Isoniazid
- Pyrazinamide
- Ethambutol
- Calculate based on the weight of the patient
- Treatment for 6 months
- 3+ (assume 50 fields)

- 0

- 1+ (assume 150 fields)

- +5 (assume 150 fields)