Sputum:

Preparation and Examination

Of Gram Stained Smears

by

Hilaire Thomas
Sputum:

• One of the most common types of specimen submitted to the

laboratory for bacterial examination.

• Difficult to obtain because of contamination with saliva.

• Even specimens collected by bronchoscopy or through an

endotracheal tube may be mixed with oropharyngeal secretions.

• Many of the bacteria which are known to cause lower respiratory tract
infections may be present in the oropharynx as part of the normal flora
e.g., staphylococci, pneumococci, and gram negative rods.

• The examination of a direct smear from the specimen can be very

helpful in diagnosing respiratory infections and in determining the
usefulness of the information provided by the culture.
The first section of this instructional unit will
show you how to prepare a smear of a
representative sample of the specimen.

In the second section we will examine the

smear and prepare a report to be recorded for
the specimen.
Equipment Required
Equipment required
The equipment needed to prepare the smears consists of
– two slides (preferably ones with frosted ends)
– a package of sterile swabs,
– a pencil
– a Bunsen burner.
Label the two slides with the patient’s name, the date of collection, and
the specimen accession number.
 Selection of the Specimen
Careful examination of the specimen will allow you to choose an appropriate
sample. The following slides will show a variety of specimen types which
may be encountered.
The specimen that is uniformly green and purulent presents no problem.
Any portion of it will provide useful information.
Specimens may be primarily clear and slightly viscid with flecks of white or
greenish material embedded. These are specimens that consists mainly of
saliva with a small amount of material that may be sputum. The flecks should
be selected.
Specimens may be heavily stained with blood. Select the portion of the sample
that is mucoid and blood stained.
When only a few blood stained flecks are present, these are the portions to select.
Avoid any part of the specimen that is watery, as this is probably saliva.
Select purulent, mucoid, or blood stained samples for examination.
Use a sterile cotton swab to extract the specimen from the container.
The sample to be used may be drawn up the side of the container with the swab to
make selection easier. Mucus can be "cut" with the swab by drawing the swab
against the side of the container and thus separating off part of the mucus. Merely
dipping the swab into the specimen will not usually provide the best sample.
 Preparation of the Smear
Place the sample on one of the labeled slides. The sample may be transferred from
the swab to the slide by a gentle rolling motion of the swab on the slide.
The sample should be about the size of a pea and should be placed near the
center of the slide.
Orient the slides so that the frosted sides are facing each other.
Hold each slide by its frosted end and place the second slide on top of the first slide
so that only the non-frosted sections are in contact with each other.
Move the slides against each other in several directions with a rotating motion. This
will spread the specimen between the slides.
To separate the slides, maintain the contact between the slides vertically and pull
the slides away from each other horizontally.
Smears of Specimen on Two Slides
If the specimen is not evenly spread on both the smears, then the slides may be
brought together again and the process repeated. This may be done several times,
if necessary, to obtain a thin, even layer of material on each slide. The finished
smears should be spread thinly and evenly over at least half of the available space
on the slide. Allow the smears to dry thoroughly before staining them.
Fix the smear by passing it through a Bunsen flame several times. Do not allow the
smear to be in contact with the flame for more than one second at a time. 5 or 6
passes through the flame is sufficient. The slide should be warm, not hot, to the
touch.
Stain one of the smears using the Gram stain procedure. Sputum
smears are usually thicker than most other types of smear and often
need more decolorization than usual. Decolorize until all or most of the
blue color is removed.
Stand the smear in a slide rack and allow the smear to air dry thoroughly.
Do not blot the smear as this may dislodge some of the material.
 Microscopic Examination
of Sputum Smears

• Begin using the low power objective so that the whole

smear can be screened. This will give a general impression
of the smear and will ensure that small areas containing
significant material will be seen.
• During the screening process, significant areas of the
smear are selected for exam under higher magnification.
• l0X or 25X objectives are suitable for screening. l00X oil
immersion objectives are used for detailed examination.
Elements Present in Sputum

• Cellular elements
• Non-cellular elements
Cellular Elements of Sputum

• Squamous epithelial cells

• Respiratory epithelial cells
• Polymorphonuclear leukocytes
• Mononuclear cells
• Alveolar macrophages
 Characteristics of
Squamous Epithelial Cells

• Large, flat, plate-like cells

• Copious clear or ground glass cytoplasm
• Relatively small dense nucleus, frequently located
eccentrically
• Sharp and clear cut cellular boundaries with
straight edges which give the cell a polygonal
appearance
• Part of the cytoplasm may be folded back on itself
• Cells are often covered with many bacteria
Squamous epithelial cells are easily seen and recognized under
low power magnification.
The lining cells of the oropharynx, their presence in a sputum
smear usually indicates contamination with saliva.
 Characteristics of
Respiratory Epithelial Cells

• Long slender cells

• Cilia at one end of the cell
• Cilia supported by basal plate
• Nucleus located at base of cell
• Slender "tail" at opposite end to cilia
 Respiratory Epithelial Cells

Respiratory epithelial cells are columnar epithelial cells which

are often ciliated. The cilia are supported by a darkly staining
basal plate. The nucleus is usually located at the base of the
cell near the tail. Epithelial cells are found in many parts of the
upper respiratory tract. They line the bronchial tree and they
are frequently seen in specimens which have been obtained
mechanically, such as bronchoscopy specimens and
transtracheal aspirations. They can also be found in the nasal
passages so that their presence is not proof of a good sputum
specimen.
Respiratory Epithelial Cells
Under low power magnification, the cells are long and slender. They are
sometimes seen in groups. They may appear spindle shaped.
 Respiratory Epithelial Cells
in Various Stages of Disintegration
Occasionally, complete cells with clearly defined cilia may be seen. More
commonly, the delicate cilia have broken off and the basal plate may or may
not be visible. Cells that have neither cilia, basal plate nor tail can be
recognized as respiratory epithelium by the oval to rectangular shape of the
cell and by the position of the nucleus which fills one end of the cell.
Occasionally almost all of the cytoplasm is gone and only the nucleus and a
small part of the cytoplasm is left. These cells are difficult to identify as
respiratory epithelium but this may be done by comparing them with less
degenerated cells in the same specimen.
 Alveolar Macrophages

• Macrophages are mobile cells which can be found in many body tissues.
• They are derived mainly from monocytes which circulate in the
bloodstream.
• They accumulate at any site of subacute or chronic inflammation. In the
transition from monocyte to macrophage, the cells become highly
phagocytic so that a macrophage is defined as a mononuclear cell which
contains phagocytosed particles.
• Alveolar macrophages are normal inhabitants of the alveolar spaces and
airways. They are very dense cells with a single nucleus which is usually
located to one side of the cell.
• The dense frothy cytoplasm often obscures the nucleus.
• The identity of the cells may be confirmed by the presence of
phagocytosed black dust particles.
• The function of these cells is to remove from the inhaled air any
remaining particulate matter which has escaped the filtration system of
the upper respiratory tract.
 Characteristics of Alveolar Macrophages

• Dense frothy or “bubbly” cytoplasm

• Nucleus located to one side of the cell – often
obscured by the cytoplasm
• Fuzzy edges of the cell due to the density of
the cell
• Presence of black dust particles or brown
hemosiderin
• Slightly orange coloration
• Synonym = dust cell
Alveolar Macrophages
Alveolar macrophages are recognized under low power by their characteristic staining
reaction and theand their size. They are very densely staining and sometimes have a
characteristic orange coloration. They are smaller than squamous epithelial cells but are
larger than polymorphs. They are often found embedded in mucus strands. The presence
of alveolar macrophages in a sputum smear indicates that the material under examination
originated from the lower respiratory tract.
The dust particles in this slide are very small and are difficult to detect. The
density of the cell is such that internal structure is not visible.
This slide also demonstrates the density of the cells. The nuclei are completely
obscured and the dust particles are clearly visible in two of the cells.
This is another low power view of alveolar macrophages. The coloration is not
quite as typical but the size and density of some of the cells is characteristic.
The frothy cytoplasm and the position of the nucleus are clearly seen in this
slide at high power magnification. Dust particles are present but are small
and difficult to see.
 Polymorphonuclear Leukocytes

• Polymorphonuclear leukocytes are commonly known as

polys or pus cells.
• The cells examined up to this point are normal residents of
the respiratory tract.
• The poly migrates into an area in response to an
inflammatory stimulus and is seen in the respiratory tract
only as a result of an acute inflammatory process.
• Frequently ,the inflammatory process is caused by bacterial
infection.
• Large numbers of polys are usually present during an acute
inflammatory reaction.
 Characteristics of
Polymorphonuclar Leukocytes

• Multi-lobed nuclei
• Granular cytoplasm
• Cytoplasm sometimes contains
phagocytosed bacteria
Polys are sometimes difficult to identify under low power. They are small cells
and their multi-lobed nuclei are usually just visible at this magnification.
At high power the intact poly is easily identified by its multi-lobed nucleus
and granular cytoplasm.
The nuclei are often more difficult to see when the cells are imbedded in mucus.
Degenerating cells are often present. The cell walls of these cells are disrupted
and the cytoplasm leaks out. The nuclei usually remain intact and the identity
of the cel1 can be established from this.
 Mononuclear Cells

• A variety of small cells with single lobed nuclei may be

present in sputum specimens.

• The Gram stain is not suitable for separating the different

types and they are therefore all grouped together as
mononuclear cells.

• Lymphocytes are the most significant members of this

group.

• Their presence is an indication of a chronic inflammatory

process, a viral infection, or certain bacterial infections.
Under low power, small mononuclear cells are difficult to identify. Their
presence can be determined under high power and relative numbers evaluated
by correlating the high power findings with the low power picture.
Under high power, any cell with a single nucleus that cannot be identified as
an epithelial cell or an alveolar macrophage is classified as a mononuclear cell.
Non-cellular Elements

• Mucus threads
• Curschmann’s spirals
• Bacteria
• Yeasts
Mucus
 Mucus

• Important part of the normal defense mechanism of the

lungs
• Produced by goblet cells which line the bronchi
• Spreads in a thin, even layer over the tissue surfaces
• Provides a transport system for the removal of foreign
bodies from the respiratory tract
• In many disease states, an excess of mucus is produced
and is expectorated
• Cells and bacteria become enmeshed in the mucus and it
is this combination of material that is known as sputum
Mucus is easily seen under low power magnification. It forms long irregularly
shaped strands which may have cells and bacteria embedded in it.
Curschmann's Spirals
Curshmann’s spirals are plugs of mucus which form in the bronchioles. Their
presence in sputum is of little significance except that they help to identify the
source of the specimen. They are easily recognized under low power magnification.
They are more resistant to decolorization than ordinary mucus and appear as
partially gram positive strands with a distinctive spiral formation.Note the close
association with mucus and cellular elements. The cells on this slide are difficult to
identify at this magnification.
This is a portion of the same spiral under oil immersion magnification.
 Bacteria

• The etiology of bacterial infections cannot be diagnosed

solely by the Gram stain morphology of the organisms
in a direct smear.

• However, the morphology of some organisms is

sufficiently characteristic that a tentative diagnosis can
be made on the basis of the smear .

• In sputum smears the most common organisms to fall

into this category are Streptococcus pneumoniae,
staphylococci, Hemophilus influenzae, members of the
family Enterobacteriaceae and some pseudomonads.
Streptococcus pneumoniae
Streptococcus pneumoniae is the most common cause of bacterial pneumonia.
Typically, the organisms are arranged in pairs and are lancet shaped. They are
frequently surrounded by a large capsule but it is usually difficult to see this on
a Gram stained smear. The capsule is seen as a halo surrounding the cells.
This will be seen more clearly on a later slide. These organisms should be
reported as lancet shaped Gram positive cocci in pairs.
Staphylococcus aureus
Staphylococci are usually much larger than Streptococci. They are usually round
or slightly oval cocci which occur singly, in anti in small clusters. Report these
organisms as Gram positive cocci in pairs and clusters. Notice that there are at
least 2 mononuclear cells amongst the polys on this slide.
Haemophilus influenzae
The presence of many very tiny pleomorphic Gram negative rods is
strongly suggestive of Hemophilus influenzae. They should be reported
as small, pleomorphic Gram negative rods.
Hemophilus influenzae often stains very weakly and tends to blend into the background
material in the smear and it is easy to overlook them. On this slide, they are most clearly
seen as intracellular organisms in the poly with a 4-lobed nucleus. Look for more
amongst the background material.
 Enterobacteriaceae
These organisms are gram negative rods but lack any more specific differential
characteristics. They are usually short fat rods, larger than Hemophilus sp.
 Pseudomonas sp.
Pseudomonads are usually long slender Gram negative rods. Like Enterobacteriaceae, their
morphology is not sufficiently typical to be able to characterize them on a Gram smear.
 Oropharyngeal Flora
A multitude of bacteria inhabit the oropharynx. A mixture of various morphologic
types, such as can be seen on this slide, is suggestive of oropharyngeal contamination.
Many of them are probably anaerobic organisms.
Yeast
Yeasts and pseudohyphal elements are frequently seen. Small numbers may be normally
present in the mouth. Increased numbers are often seen in patients who are immuno-
logically compromised and in those who have been extensively treated with antibiotics.
These patients often have oral candidiasis and yeasts are seen in association with squamous
epithelial cells. Association with mucus and polys may represent pulmonary colonization in
these patients. Yeasts are much larger than bacteria. Notice the size difference between the
yeast and the gram negative rods.
Evaluation of Specimen
The presence of alveolar macrophages provides positive evidence that the specimen
originated from the lower respiratory tract. These cells will be detected under low
power. The cells in this slide are mostly alveolar macrophages. Mucus is also present.
The orange coloration of the alveolar macrophages can be easily seen on
this slide. Mucus, polys, and other mononuclear cells are also present.
Mucus is another indicator that sputum is present By itself, it does not pro-
vide adequate proof that this is so. Curschmann's spirals are only produced in
the lungs Therefore, if these are seen, the presence of sputum is confirmed.
The presence of large numbers of polys with mucus, even in the absence of alveolar
macrophages, is good evidence of the presence of sputum especially if there is a
single morphologic type of bacteria present. Alveolar macrophages are frequently
absent in extremely purulent specimens.
Squamous epithelial cells originate in the mouth. They are usually seen with many bacteria
of varying morphologies. Specimens that consist solely or predominantly of squamous cells
and mixed flora are considered inadequate and represent specimens of saliva only.
• Unfortunately, most specimens fall somewhere
between these extremes

• Many, or all of the elements mentioned are

present in varying quantities.

• Sometimes, although there is evidence that saliva

is present, there may be some areas of the smear
that do adequately represent sputum.
This slide shows polys, alveolar macrophages and one contaminating squamous
cell. This would be considered a good specimen, since contamination is minimal.
This slide shows a predominance of oropharyngeal material but there are also many
polys present. This should be reported as an inadequate specimen with the presence of
polys noted. Specimens that show an even mixture of sputum and saliva are reposed as
for sputum but with the notation that the specimen was contaminated with saliva.
The presence of respiratory epithelial cells is of little help in establishing the
nature of the specimen. They are usually present in bronchoscopy specimens.
Reporting
Report the presence or absence of alveolar macrophages
for all specimens.

Report either:
“Alveolar macrophages present“,
or
“No alveolar macrophages seen”.
Report mucus only when it is present.

Report:
“Mucus present”.
Polys, mononuclear cells, and epithelial cells are
reported with an approximate quantitative value.
The average number of each type of cell per low
power field in a representative area of the smear
is recorded.
 Quantitation of Cells

• The average number of each type of cell per low power

field in a representative area of the smear is recorded.

• Less than 1 and up to 9 cells per field are reported as

“Few”

• 10 to 25 cells are reported as “Moderate”

• Any number in excess of 25 is reported as “Many”

 Quantitating Bacteria

• Terms are the same as those used for cells

• Organisms are counted using the oil immersion objective

• Organisms that are seen between once in every ten fields and
once in every field are reported as “Few”

• More than one organism per field but less than 50 per field is
reported as “Moderate”

• Greater than 50 organisms per field is considered "Many"

• Bacterial types are reported individually when their
presence suggests that they may be the cause of an
infectious process.

• They are reported by Gram morphology when not

more than two morphologic types are present or
when there is a single type which predominates over
the mixture of other organisms.

• In order to be considered a single morphologic type

of organism, the morphologic appearance of all
members of the population must be consistent with
a single genus or species.
This slide represents mixture of Hemophilus and pneumococci. If this slide represented
the whole field, the pneumococci would be reported as moderate and the Hemophilus as
heavy. Notice the halo effect of the capsule around the pneumococci and the light
staining of the Hemophilus.
When multiple morphologic types are present they are reported as "Mixed Organisms"
and are considered as one morphologic type for quantitation purposes. This slide shows
heavy contamination with oropharyngeal flora including Yeasts. Notice the indistinct
nuclei of these polys.
 Descriptive Terms for Bacteria

Qualifying descriptive terms can be used if the bacterial

morphology is sufficiently typical. The most commonly used
terms are:

• “Gram positive cocci in clusters” representing staphylococci.

• “Gram positive cocci in pairs and chains” representing
streptococci.
• “Gram positive cocci, lancet shaped” representing
Streptococcus pneumoniae.
• “Gram negative bacilli, small pleomorphic” representing
Hemophilus sp.

Organisms should never be identified more specifically from a

Gram smear.
You will recognize these as probable staphylococci.
You should recognize these as a mixture of pneumococci and Hemophilus.
streptococci other than Streptococcus pneumoniae are usually more spherical and
chains of at least four organisms should be seen. These organisms are not often seen
as causes of pneumonia.
 Other Descriptive Terms

• “Specimen inadequate. Saliva only”

• “Repeat requested”
• “Squamous epithelial cells only”

These terms are used together and may

constitute the whole report.
Terms in previous slide are used when only squamous epithelial cells and
mixed organisms are present as in this example.
 “Suggests contamination with saliva”

This term is used when one or more of the elements

representing sputum is present but squamous
epithelial cells and mixed flora are also present in
heavy amounts.
If many squamous epithelial cells and some polys are present, report these cells
with quantities and use the descriptive terms also. This smear would be reported
as moderate polys, many squamous epithelial cells, specimen inadequate, repeat
requested.
 “Scanty specimen”

This term is used when there is little

cellular material to be examined. This
type of specimen is usually inadequate
for interpretation.
 Read and Report Sputum Gram Smears
Summary of Steps

• Scan the whole smear to determine whether sputum is

present and to form general impression as to the nature of
the specimen. Look especially for alveolar macrophages,
mucus, Curschmann's spirals, polys and epithelial cells.
• Pick an area that seems to represent sputum and examine
under oil immersion. Identify the cells present and look for
potentially pathogenic organisms. Identify the organisms
by Gram morphology and estimate the numbers present.
• Return to low power and quantitate the cellular elements.
• Write the report.
 Segments of Report

• Presence or absence of alveolar macrophages.

• Presence of mucus.
• Nature and frequency of other cells
• Nature and frequency of bacteria
• Descriptive phrases for the closer identification of
bacteria and any other appropriate phrases. In
cases where the specimen consists of only saliva,
then just the descriptive phrases are used.
There are many variables involved in the
examination of sputum smears. These range
from the nature of the specimen itself to the
way that the smear was made and stained. The
prejudice of the examiner will also influence
the report to some extent.
This module has described the common
components which may be seen in sputum
smears and should provide the basic
information needed for the student to begin
to look at sputum smears with confidence.