MT 111 LECTURE 4: MICROSCOPIC EXAMINATION METHODS

OBJECTIVES:

 Understand the importance of microscopic examination of infected materials.

 Know the technique in the preparation of samples from different sources for microbiology laboratory examination.
 Explain the purpose of staining microorganisms.
 List the procedure in doing Gram staining and describe the appearance of gram-positive and gram-negative organisms.
 Know the procedure of acid-fast staining
 Understand the special staining to employ for the demonstration of special structures
 Microscopic Examination Methods are laboratory procedures/tests that directly visualizes the contents of a clinical
specimen.
 Clinical is attributed to disease state/infection/condition of a certain patient whereas specimen can be anything taken out
from a patient (blood or any body fluids) that is sent to the laboratory for examination.
 Clinical specimens are samples taken from the body of the patient that is sent to the laboratory for examination so that a
diagnosis can be made from, the disease state/infection/condition state of the patient can be established by the physician.
 Microscopic Examination- methods of direct visualization of microorganisms under the microscope
o It is submitting the clinical specimen of the patient to microscopic viewing to see the individual makeup/component of the
clinical specimen. These individual components of the clinical specimen is not visible to the naked eye and is a need to use
microscope to view it. These components might tell an information to the physician that the condition/disease state of the
patient must be diagnosed in this particular manner.
 The clinical laboratory has three phases of workflow, and post analytical phase.
o Pre-analytical phase (most critical)- involves the collection of the specimen, the laboratory receiving the specimen and
checking its integrity.
 Included are the instructions from the laboratory/laboratory technologist on how the patient should collect the sample
(when certain tests will entail that the patient themselves will collect the sample) so that the analytical phase is correct as
well as the results in the post-analytical phase
o Analytical phase- involves the examination of the specimen
o Post analytical phase- when the laboratory sends the report/result to the physician for reading and interpretation
 The importance for the direct visualization of the contents of the clinical specimen is that we want to establish the
presence/absence of the disease state in that particular patient. In microbiology, the microscopic examination methods
evolved in the context of microscopic examination of infected materials. Infected specimens are specimens which has
microorganisms in it causing the disease state to the patient. The importance for the microscopic examination of infected
materials is to establish the presence of microorganisms in the clinical specimen. If the results from the laboratory (given to
the physician) have established that there is presence of microorganism that will confirm the disease state of the patient. On
the other hand, when the report from the laboratory stated that there is no presence of microorganisms in the clinical
specimen, it will refute the idea of the physician of the disease state of the patient and that will give the physician a
challenging instance of additional investigations so that his/her investigation will be lead to another route on how the
physician can lead to a correct detail so that the disease state could be confirmed and correctly diagnosed.
 The main core of microbiology lab examination is the identification of the organism causing disease to the patient. One
procedure is microscopic examination for presumptive identification of the organism which is an initial identification of the
organism. Microorganisms are stained during microscopic examination for us to presumptively identify the morphology of the
organism. Gram staining procedure will divide two different types of organism: gram (+) which appears violet/purple/blue,
and gram (-) which appears red/pink. If the laboratory technologist have viewed a purple-colored microorganism, that
laboratory technologist will have a presumptive identification that the organism is gram (+) and that will help the
investigation in the microbiology laboratory narrow down to the potential gram (+) organisms that can cause infection to the
specimen.
 Morphology encompasses 4 aspects: size, color, shape, and arrangement. These 4 aspects are what we look at to
presumptively identify the organism.
PREPARATION OF SAMPLES

 How the microbiology laboratory prepares samples for microscopic examination ensuring that the pre-analytical phase is
accurate so that in turn the analytical and the post-analytical phases in microbiology laboratory also gets a correct and
accurate result.
A. Smears from swabs
 Cotton swabs is like cotton buds. An applicator stick is applied with cotton at the tip and it is tolled at the tip of the
applicator stick and that is already a cotton swab.
o Cotton swabs made in the laboratory are sterilized, so after a cotton swab was made, all the cotton swabs are submitted
to autoclave machine to sterilize it and that is now ready to collect samples from a certain patient.
o In order to prepare this sample for microscopic examination, we need to transfer the absorbed material from the cotton
swab into a clean microscope slide so that it will facilitate easy microscopic examination of that infected and absorbed
material from the cotton swab.
 Microscope glass slides
o The demarcation line in the microscope slide is telling you that this area is the frosted end of the glass slide. If you use the
lead of the pencil, if you scratch the lead of the pencil in the frosted end which is a rough end of the glass slide, the lead
of the pencil will leave markings in it. The entire side of the glass slide where the frosted end is, is called frosted side of
the glass slide. The opposite side is called non-frosted side.
o In order for us to facilitate a microscopic examination of the collected bacteria in the cotton swab, we need to transfer it
to the frosted side of the glass slide because the frosted end of the glass slide should contain the patient details so that
correct label of that specimen is established and the results of that correctly labeled slide is released to the correct
patient also.
o Patient details we need to place in the frosted end of the glass slide:
 Patient’s name (family name first then first name), birthdate of the patient, sex, date and time of collection of the
sample and the specimen source.
 Date and time of collection is important because if a specimen is collected from a cotton swab, the cotton swab is
transported to the laboratory for microscopic examination and we cannot process specimens collected from cotton
swabs at a later time because that will cause drying up of the absorbed material in the cotton swab. After a specimen is
collected from a cotton swab, it should be made into smear right away just before the material gets totally dried up in
the cotton swab to get a yield and a correct representation of that specimen collected from that cotton swab.
 Procedure:
o Once the cotton swab is transported to microbiology laboratory, transfer the absorbed material from the cotton swab to
the frosted side by rolling the cotton swab all over the surface of the clean glass slide because rolling the cotton swab will
transfer all the absorbed material from the cotton swab to the surface of the glass slide and in that sense, we are
transferring a correct representation/the entire representation of the collected material from the cotton swab of that
particular patient.
 It is not advisable to rub the cotton swab back and forth in the surface of the glass slide because rubbing the cotton swab
back and forth in the surface of the glass slide does not transfer the entire collected material from the cotton swab because
only one side of the cotton swab is in contact with the surface of the glass slide and the other side of the glass of the cotton
swab is not transferred and is not in contact to the surface of the glass side so we are not getting a correct representation of
the clinical specimen.
 If samples can be collected only on swabs, and we are about to prepare smears for microscopic examination, there should
be two different smears from the same patient and same specimen source to establish consistency in the contents of the
clinical specimen being examined. If there are two cotton swabs sent to the lab collected from one patient from the same
specimen source, two different smears have to be made.
 Once both smears made from the two cotton swabs are completed and stained, once the findings in the first smear
revealed that Staphylococcus aureus is present in the specimen causing the disease to the patient, the second smear which
came from the same patient and specimen source should also reveal Staphylococcus aureus to be consistent in the content
 of that same specimen. When there is consistency and the data is congruent to each other, the result can be released to the
physician that this is the microorganism of interest that is to be treated in order for the patient to recover from the disease
state.
 When one smear revealed Staphylococcus aureus and the other smear revealed Escherichia coli, the discrepant and
inconsistent data is a ground for recollection of sample because such results cannot be released to the physician because of
two different organisms in the different smear yet coming from the same patient and source.
B. Smears from thick liquids or semisolids
 Thick liquids are viscous, very sticky specimens (sputum/phlegm); semisolid (feces or stool)
 Thick liquids or semisolids are not directly collected by laboratory technologists as opposed to cotton swabs. These are
collected by the patient and these specimens are collected in a specific sterile container.
 When thick liquids or semi-solids are the specimen of choice to rule in a specific disease state of the patient, then
instructions from the laboratory should be clear and understood correctly by the patient so that these specimens are
collected correctly.
 Procedure:
o If the thick liquids or semisolids are collected in a container and the container is sent to microbiology laboratory for the
microscopic examination, then make smears from the collected thick liquid or semi-solid in a container.
o By the use of cotton swab, immerse the cotton swab into the container and allow the cotton swab to absorb the
specimen in the container. After the cotton swab has absorbed the correct amount of sample needed to be transferred
into a glass slide, the cotton swab is rolled all over the surface of the glass slide to transfer the entire absorbed material
from the cotton swab to the surface of the glass slide and the glass side should be properly labeled.
o Do not only settle to only one cotton swab on one smear. Use a second swab, immerse it again and make a second smear
from the second cotton swab and the purpose is to establish consistency in the content of the result of that clinical
sample.
C. Smears from thick, granular, or mucoid materials
 Thick, granular, or mucoid materials is same with thick liquids which are sputum specimens but differ in the
granules/granular material in it.
 If the sputum that the patient expectorated has granules/granular material, it is an indication of something abnormal
because normal sputum collection does not involve the production of granules/granular material.
 Procedure:
o If there are granules/granular material collected together with the sputum specimen from the patient, that is a possible
specimen of interest that we want to study and reveal its contents because it might be the answer as to why the patient
submitted him/herself to laboratory tests. First thing to do is to make a smear from that granular material.
o Fish out/remove the granules from the container and place it in the center of a clean, properly labeled glass slide. The
ideal way to make smear from granular material is to crush the granular material and make a smear out of it.
o After fishing out/remove the granular material from the specimen container, place it in a clean, properly labeled glass
slide using a second clean, properly labeled glass slide with the frosted side facing down. The second glass slide is
positioned on top of the first glass slide where the granules are placed and push the second glass slide down to initially
crush the granules in between the two glass slides.
o After initial crushing of the granules, the two glass slides are rotated against each other, rotated at different direction
because the extra pressure in rotating the two glass slides against each other will completely crush the material in
between them.
o After the granules are completely crushed up within the two glass slides, they are pulled away creating two different
smears from one granular material to establish consistent results or contents in the granular material that a smear was
made from.
D. Smears from thin fluids
 Thin fluids are specimens with low protein content and low cellular count while thick fluids are specimens with high
protein content and high cellular count.
 Staining specimens for microscopic examination will reveal the true content/makeup of the clinical specimen. Staining a
smear from a clinical specimen would help the laboratory technologists in establishing the disease state of the patient.
 Smearing
 o If the specimen received in the laboratory contains high protein level and the cellular elements are also very numerous,
when that specimen is smeared in a clean glass slide, when submitted to staining and after the staining process, apparent
visual colors are seen in the surface of the glass slide because it is the high protein content and high cellular content of
that specimen that takes up the stain making it visible after the staining process. Since there is visible colors in the surface
of the glass slide, the laboratory technologists microscopic examination will be easily facilitated because the drop of oil
will be placed on the visible colors of the glass slide surface.
o When thin fluids are smeared and spread in the surface of a clean glass slide, after the staining process, remnants of
visible colors are not seen because it has low protein content and cellular elements. The laboratory technologists will have
a hard time in doing the microscopic examination because they have to search in the entire glass slide surface where the
smear was which will affect the turnaround time of the laboratory test and the diagnosis of the patient is not done in a
timely manner.
 Procedure:
 Instead of spreading the thin fluid in the entire surface of the glass slide, to make a smear from thin fluids, place a drop
in the center of the frosted side of the glass slide which was pre-marked on its non-frosted side of the glass slide.
 Glass slides are pre marked using a wax pencil, the non-frosted side is marked with a circle shape
 After making the smear, air dry and stain it. If there is no visible colors seen in the glass slide surface, it will be easy for
the laboratory technologist to locate where the smear was because he/she may locate within the perimeter of the
circular shape and will not affect much of the turn-around-time.
 Cytocentrifuge
o The best and most recommended way of preparing smears. Spinning the cells to concentrate and sediment them in the
test tube.
o Procedure:
 Small aliquots of the thin fluid (0.1 to 0.2 mL) are placed a test tube and submitted to centrifugation/spinning process
and the material is spun for 10 minutes. If that thin fluid contains low cellular and protein content, after the spinning
process, all the cellular elements in the protein content gets sedimented and concentrated at the bottom of the test
tube. Since the cellular element and the protein content are the substances of interest in the microscopic examination,
the liquid portion of the specimen after the spinning process is discarded.
 The sediment is aspirated using a pipette and a drop of the concentrated specimen is placed in the center of a clean,
properly labeled glass slide (frosted side of the glass slide). It is fixed and decontaminated in 70% alcohol for 5 minutes.
 Since it is concentrated material, it cannot be submitted to staining because it is difficult for the laboratory technologists
to view the morphology of the cells in a thick, concentrated material so a thin material has to be made from the thick
material using a wire loop.
 From the wire loop, emulsify the thick material and make a thin smear by bringing/making a swift motion to the right
creating a thin portion. The slide with thick and thin portion is submitted to staining and visible colors can be seen and
do the examination at the thin portion.

PREPARING INFECTED MATERIALS FOR VISUAL EXAMINATION

Preparation Specimen or Organism type

For gross examination:
 Wet preparation Parasites; Materials >1 mm in size
For microscopic examination:
 Wet preparation (direct or Fluids or semisolids
sedimented)
 Cytocentrifuge Clear or slightly turbid fluids
 Smear Clear of slightly turbid fluids
1. Drop Pus or fluid; Tissue homogenate; Swab rinse
2. Pellet Blood culture; Direct specimen
 3. Rolled Swabbed material
4. Imprint (touch preparation) Tissue
 Before staining the smears, two prior processes needed to be done completely
1. Air drying- in air drying the smears, there is no particular time to follow in air drying smears because it will depend on the
thickness of the smear. If a certain time is followed in air drying the smear, the result is prematurely air dried smear (portion
of the smear is still wet and if submitted to staining, that wet portion gets washed out in the staining process and that
washed out area might contain the more important findings of the specimen so risks cannot be taken)
o If the smear is too thick, 10-15 mins (of some textbooks) may not be enough or if the smear is too thin, 3 mins (of some
textbooks) may be too much. Grossly view the smear and decide on our own if it is completely air dried already.
o Air drying will preserve the morphology of the organism.
2. Heat-fixing- exposing the non-frosted side of the glass slide to the flame of an alcohol lamp for several times and it
simultaneously kills the microorganism and it also adheres the smear into the slide (will not get washed out during the
staining process)
 The two prior processes should be done completely so that the staining process is done correctly

STAINING OF SMEARS

 Coloring the microorganisms with a dye in order for the medical technologist to presumptively identify the morphology of the
organism, helping the microbiology laboratory narrow down the investigation to the potential/possible organisms causing
infection to that clinical specimen.
 There are 4 theories/principles involved in staining of smears
1. Stains are salts composed of two ions, positive ion and negative ion
2. One of the two ions in the staining solution successfully/effectively colors the microorganism (chromophore)
3. The positive ion in the staining solution is carrying the colors of the basic dye whereas the negative ion in the staining
solution is carrying the colors of the acidic dye
4. The cell wall of microorganisms at pH 7 is slightly negative
o The four principles stated that staining solutions contain two ions, positive ion and negative ion and one of these ion stains
the microorganism effectively and from that ion, it is called chromophore. The positive ions in the staining solution has the
colors of the basic dyes whereas the negative ions has the colors of the acidic dyes. Since the cell wall of any
microorganisms at pH 7 is slightly negative and the staining solutions are composed of positive and negative ion, therefore
the positive ion in the staining solution gets attracted to the cell wall of the microorganism because the cell wall is slightly
negative.
o As the positive ion gets attracted to the cell wall of the microorganism because it is slightly negative, the positive ion or the
colors of the basic dyes will color the microorganisms’ cell wall. Therefore, the positive ion is the chromophore.
o The colors of the acidic dyes which are in the negative ion, since the negative ion repels the cell wall of the microorganism,
they color the background of the organism and that is also very relevant and important in staining microorganisms because
in order for the microbiology laboratory to do a presumptive identification of the organism, the organism’s color should
have a contrasting background so that the microorganism can be seen and viewed properly under the microscope.

THREE KINDS OF STAINING TECHNIQUE IN MICROBIOLOGY LABORATORY

 Simple stain
o
Is an aqueous or alcohol solution of a single basic (alkaline) dye.
o
It is not used in staining microorganisms because the morphology of the microorganisms can never be established since
simple staining makes use of only one colored stain so whatever the color of that stain, that will be the color of the
organism and also its background.
o Sometimes an additive known as mordant is added to the solution
o Directed toward coloring the forms and shapes present
o Methylene blue, crystal violet, carbolfuchsin, and safranin
 Differential stain
o It is not termed as differential because it employs more than one stain. It is termed as differential because it differentiates
microorganisms by its color.
o Employs more than one solution of dye
o React differently to different kinds of bacteria
o Directed toward coloring specific components of the organism
o Gram Stain (Hucker’s method)
 If microorganisms are stained using gram staining procedure, the lab can immediately separate Gram (+) organisms from
Gram (-) organisms by taking into consideration the color reaction of the organism.
 Developed by Hans Christian Gram
 Fastest and least expensive method for presumptive diagnosis in clinical settings
 Procedure:
1. Make a thin smear.
2. Flood the smear with crystal violet for 1 minute. Then wash with tap water.
3. Cover the smear with Gram’s iodine for 1 minute. Then wash with tap water.
4. Decolorize the smear with acetone alcohol until a faint violet color flows off the slide.
5. Counterstain with safranin for 30 seconds. Then wash with tap water.
6. Dry and examine under OIO.
 Gram (+) organisms appears as violet/pink/blue because it has a cell wall composed of thick peptidoglycan layer with
teichoic acid which are insoluble in alcohol and are made up of disaccharides and amino acids (rich in protein).
 Gram (-) organisms appears as red/pink because the cell wall is composed of thin peptidoglycan layer with
lipopolysaccharides which are soluble in alcohol and are made up of lipids and polysaccharides.
 The solubility of the cell wall of these organisms to alcohol is in the context of which cell wall is destroyed by alcohol.
Since teichoic acid (Gram (+) cell wall) is insoluble in alcohol, that means the cell wall of Gram (+) organisms are not
destroyed by alcohol and since lipopolysaccharides are soluble in alcohol, that means the cell wall of Gram (-) organisms
are easily destroyed by alcohol.
 It involves 4 steps:
1. Primary staining
 It involves the application of a primary stain called crystal violet which is a violet-colored stain with the chemical name
hexamethyl-para-rosaniline chloride.
 Gentian violet is an alternative to crystal violet when crystal violet is not available in the microbiology lab.
 Both gram (+) organisms (represented by round organisms) and
gram (-) organisms (represented by rod-shaped organisms) appear
violet because that is the only colored stain that was employed
yet to the organism
 60 seconds.
2. Mordant
 Gram’s iodine is the reagent used, brown-colored reagent
 It will intensify the color of the primary stain to the cell wall of the
microorganism. The mordant will bridge more of the color of the
primary stain to the cell wall of the microorganism so that the microorganism receives a darker color of the primary
stain.
 At the end of the mordant step, both gram (+) and gram (-) organisms still appear violet (darker violet in color).
3. Decolorization/differentiation
 It makes use of a decolorizer called acetone alcohol which is basically a combination of acetone and alcohol.
 Crucial step in the gram staining procedure because gram (+) and gram (-) organisms react differently to the reagent.
 If gram (+) organisms are exposed to acetone alcohol, the cell wall of gram (+) organisms will remain intact and will
never get destroyed by the alcohol’s action as a decolorizer. Since the cell wall of gram (+) organisms are not
destroyed by acetone alcohol, the crystal violet color is retained inside the cell wall of gram (+) organisms, that is
why they still appear purple/violet after decolorization step.
  If gram (-) organisms are exposed to acetone alcohol, because of its lipopolysaccharide content and its thin cell wall,
the lipopolysaccharides in the cell wall of gram (-) organisms gets destroyed. Acetone alcohol penetrates the cell wall
of the gram (-) organism and decolorizes the crystal violet in the cell wall and will leave the gram (-) organisms
colorless after decolorization step.
 This step is called decolorization because it decolorizes gram (-) organisms. It is called differentiation because it
differentiates gram (+) organisms from gram (-) organisms according to their reaction to the acetone
alcohol/decolorizer.
4. Counter staining/secondary staining
 It makes use of a counter stain called safranin which is a red-colored stain.
 Since gram (+) organisms from the decolorization process retains the purple/violet color then flooding gram (+)
organisms with safranin will no longer take up its red color and after the gram staining procedure, it will remain
purple.
 Since gram (-) organisms from the decolorization process became colorless then flooding gram (-) organisms with
safranin, they will take up the safranin and its red color and after the gram staining procedure, they appear red.
 Reason for the appearance of gram (+) organisms under the microscope: thick peptidoglycan layer with teichoic acid
 Reason for the appearance of gram (-) organisms under the microscope: thin peptidoglycan layer with lipopolysaccharides

PRINCIPLES OF STAINING (GRAM-POSITIVE ORGANISMS)

 Heat-fixing creates pores/openings in its cell wall as it gets exposed to the flame.
 Crystal Violet
 Since its cell wall has pores/openings from the heat fixing step, the crystal violet
can easily enter the cell wall of gram (+) organisms so that at the end of the
primary staining, gram (+) organisms will appear purple.
 Crystal violet is a smaller molecule compared to the size of its pores/openings in its
cell wall.
 Gram’s Iodine
 The iodine molecule also can easily penetrate the cell wall of gram (+) organisms
via its pores/openings
 Once iodine molecule have entered the cell wall, the iodine molecule will form an attachment with crystal violet
molecule already inside the cell wall of gram (+) organisms creating/forming a complex called crystal violet iodine
complex (CV-I complex)
 The size of the CV-I complex is bigger than the size of the pores/openings in the cell wall of the gram (+) organism. If we
try to decolorize the CV-I complex in the cell wall of gram (+) organisms, the CV-I complex cannot escape anymore from
the cell wall of the gram (+) organisms because its size is bigger than the size of the pores/openings in the cell wall of
the gram (+) organism.
 Acetone Alcohol
 Since teichoic acids are made up of disaccharides and amino acids (proteins), once the gram (+) organisms are exposed
to acetone alcohol, it should decolorize the CV-I complex from the cell wall of gram (+) organisms but in a different
reaction because of the teichoic acid in the cell wall of gram (+) organisms.
 Since it is richly composed of proteins, alcohol will coagulate/denaturate the proteins in the cell wall of gram (+)
organisms. Instead of creating bigger openings, it will coagulate and harden the proteins causing the teichoic acid to
close the openings/pores from its cell wall brought about from the heat-fixing step. The CV-I complex gets trapped
permanently in the cell wall of the microorganism. After the decolorization step, gram (+) organisms are still purple in
color.
 Safranin
 Since from the decolorization step, the cell wall of gram (+) organisms pores/openings in its cell wall has already closed
because the teichoic acid coagulated with alcohol because it is not destroyed by alcohol. The CV-I complex gets trapped
within the cell wall of the gram (+) organisms.
 When flooded with safranin, safranin molecules will only stick or attach initially or temporarily on the coagulated area of
its cell wall and since it only attaches, its attachment is very loose and is not stable, after safranin in staining. The entire
 smear is submitted to washing with tap water. Once the entire gram (+) organism with safranin molecule initially
attached only in the coagulated area to tap water, the tap water washing will wash out the safranin molecule that is
attached leaving gram (+) organisms completely violet/purple.

PRINCIPLES OF STAINING (GRAM-NEGATIVE ORGANISMS)

 The heat-fixing step will also create pores/openings in the cell wall of gram (-) organisms.
 Crystal Violet
 Once crystal violet is added, it can immediately enter the cell wall of gram (-) organisms and after the primary staining
step, gram (-) organisms appear purple/violet.
 Gram’s Iodine
 The iodine enters its cell walls via the openings/pores and form a complex with crystal violet molecule producing the
crystal violet iodine complex (CV-I complex) molecule which is bigger than the size of the pores/openings in the cell of
gram (-) organisms and so it will not be washed out. Gram (-) organisms still appears purple/violet.
 Acetone Alcohol
 The gram (-) organism reacts differently from that of the gram (+) organisms.
 Since its cell wall is composed of lipopolysaccharides, as soon as gram (+) organisms are flooded with acetone alcohol,
once the alcohol touches the cell wall of gram (-) organisms, the cell wall of the gram (-) organisms gets destroyed and
the resulting product is it creates an even bigger opening in its cell wall and the size of the pores/openings in the cell
wall of gram (-) organisms after the alcohol destroyed its lipopolysaccharides is bigger than the size of the CV-I complex
inside.
 As the alcohol touches the entire gram (-) organism, the CV-I complex can go out/escape the cell wall of gram (-)
organisms leaving gram (-) organisms colorless after the decolorization step.
 Safranin
 Since the cell wall of gram (-) organisms now has bigger openings from the destruction of alcohol of its
lipopolysaccharide content, the safranin molecule can freely enter the bigger openings in its cell wall and will stain the
gram (-) organisms red.
 When washing the gram (-) organism, the safranin molecule will not be washed away since the safranin molecule has
entered the cell wall successfully. In the end of the entire gram staining procedure, gram (-) organisms will appear red.

ACID-FAST STAIN

 Is a staining technique that differentiates acid-fast organisms from non-acid-fast organisms in terms of its color reaction to
the staining process.
 Procedure:
 Ziehl-Neelsen method
1. Prepare smears of sputum.
2. Apply carbolfuchsin and bring to steaming by holding the slide above a
small flame.
3. Decolorize with acid alcohol.
4. Counterstain with Loeffler’s methylene blue for 30-45 seconds. Then wash
with tap water.
5. Dry and examine under OIO.
 Kinyoun staining method
1. Prepare smears of sputum.
2. Stain with Kinyoun carbolfuchsin for 3 minutes. DO NOT heat. Rinse with water.
3. Decolorize with acid alcohol.
4. Rinse with water and counterstain with methylene blue for 30 seconds.
5. Rinse with water and air dry.
6. Examine under OIO.
 Acid-fast organisms appears red against a blue background whereas non acid-fast organisms appears blue.
  Example: Mycobacterium tuberculosis (causative agent for pulmonary tuberculosis)- has metachromatic granules (much
granules), Mycobacterium leprae, Nocardia organisms (partially acid fast)
 Two methods used
1. Ziehl-Neelsen method (hot method)- Involves steaming/heating the smear in the process
2. Kinyoun method (cold method)- Does not involve heating/steaming. It increases the concentration of the phenol so that
the acid fast organisms are stained effectively.

PRINCIPLES OF STAINING (ACID-FAST ORGANISMS)

 The cell wall of acid-fast organisms is composed primarily of mycolic acid which are fatty acids that makes the cell wall of
acid-fast organisms very slippery/slimy. Other name: hydroxymethoxyl acid
 Mycolic acid is responsible for the acid-fastness of any acid-fast organism.
 Ziehl-Neelsen method involves 4 steps:
1. Primary staining (Carbol fuchsin)
 It involves the application of a primary stain, carbol fuchsin, a red-colored dye
 The cell wall of acid-fast organisms are rich in mycolic acid which are fatty acids.
 For acid-fast organisms, the heat-fixing will cause pores/openings in the cell wall of acid-fast organisms because the
heat will cause mycolic acid in its cell wall to relapse.
 After heat-fixing the smears for Ziehl-Neelsen procedure, it was allowed to cool down. No pores/openings are seen
since cooling down the smear before submitting it to staining for acid-fast staining procedure will cause the mycolic
acid to close again. It causes the carbol fuchsin molecule to just stick initially on its outside cell wall leaving acid-fast
organisms after the primary staining colorless.
2. Mordant (Steaming)
 The application of a flame from an alcohol lamp to the non-frosted side of the glass slide and expose the entire smear
to the flame for a specific time until a steam comes off the slide (steam only, do not boil)
 Since the cell wall of acid-fast organisms are exposed to heat, the mycolic acid again relapses and will create
pores/openings in its cell wall. Since the carbol fuchsin molecules were initially attached from the primary staining
step, as soon as it opens the cell wall when mycolic acid relapses, the carbol fuchsin molecule can immediately enter
its cell wall coloring acid-fast organisms red.
 After steaming or application of heat, the smear was allowed to cool down. In the cooling process of the smear, the
carbol fuchsin already had penetrated the cell wall of the acid-fast organism. The cooling process will lock again the
pores/openings in the cell wall of the acid-fast organism because mycolic acid closes it again, causing carbol fuchsin
molecule to be trapped inside the cell wall of acid-fast organisms permanently.
3. Decolorization (Acid alcohol)
 The acid alcohol molecule will only attach initially on the outside cell wall of the acid-fast organisms. Acid alcohol will
just slides off the cell wall of acid-fast organisms because it is slimy and slippery because of its fatty acid content
 The acid alcohol will just be washed out and will leave the acid-fast organism not decolorized and retain its red color.
4. Counter staining/secondary staining (Loeffler’s alkaline methylene blue)
 Since the cell wall of acid-fast organisms are no longer opened, the LAMB molecule will also initially attach to the
outside cell wall of the acid fast organisms and will eventually slide off the cell wall because of its slippery/slimy nature
and the acid-fast organism is still red at the end of the Ziehl-Neelsen procedure.

PRINCIPLES OF STAINING (NON-ACID-FAST ORGANISMS)

 The cell wall of non-acid fast organisms does not contain mycolic acid and when submitted to heat fixing, it will create
pores/openings in its cell wall because of the lack of mycolic acid.
1. Primary staining (Carbol fuchsin)
 Carbol fuchsin molecule can easily penetrate the cell wall of non-acid fast organisms because of its pores/openings
leaving non-acid-fast organisms after the first step colored red.
2. Mordant (Steaming)
  In the steaming/heating process, the pores/openings in the cell wall of non-acid-fast organisms becomes even bigger
because of heat and leaving non-acid-fast organisms colored red because alcohol is not applied yet.
3. Decolorization (Acid Alcohol)
 Since the cell wall of these organisms contain bigger pores/openings from the heating/steaming process, the acid
alcohol molecule can easily enter the big pores/openings so that it can decolorize the carbol fuchsin and carbol fuchsin
will escape the cell wall of non-acid-fast organisms leaving non-acid-fast organisms colorless after decolorization.
4. Counter staining/secondary staining (Loeffler’s alkaline methylene blue)
 The LAMB molecule can easily penetrate the cell wall of non-acid-fast organisms and therefore colors them blue at the
end of the staining process.

SPECIAL STAINS

o Used to color and isolate specific parts of the organism

o Demonstrate the presence of spores, capsules, flagella, and metachromatic granules which are responsible for the virulence
o Some stains used to demonstrate different bacterial structures:
 Writz’s staining for spores
 Modified Maneval’s for capsule staining
 Leifson’s method for flagellar staining
 Albert stain, Loeffler’s methylene blue or Gram stain for metachromatic granules
o Capsules and slime layer- unstained structures surrounding the microorganisms which vary in amount. If the unstained
lipopolysaccharides are abundant, it is a capsule but if it is scanty, it is a slime layer.
 Virulence mechanisms: anti-phagocytosis and anti-complimentary (capsule); attachment/adhesion (slime layer)
o Flagella- thin structures extending from the body of the microorganism
 Virulence mechanisms: motility and antigenic property
 MESSEA’s classification: flagella have 4 different types according to the number of flagellum/flagella and its location within
the body of the organism
o Spore/endospore- contains calcium dipicolinate/dipicolinic acid;
 Virulence mechanisms: resists disinfection
 Different types depending on its location: central, sub-terminal (Bacillus subtilis), terminal (Clostridium tetani)
o Metachromatic granules- demonstrates metachromasia; Corynebacterium diphtheriae, Mycobacterium tuberculosis
 Virulence mechanism: energy reserve

REPORT OF DIRECT EXAMINATION

 After microorganisms are stained, they are now ready to view microscopically and to possibly report the results to the
physician
 It should be simple and include all information elements needed by the physician
 Details that needed to be established in the report:
o Date and time of specimen receipt
o Specimen type or source of specimen
o Microscopic findings (Organism morphology and Background material)
o Date and time of testing completion
o Requesting physician
o Medical technologist and signature- result will not be considered valid and accurate if not present