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Sputum: Preparation and Examination Of Gram Stained Smears by Hilaire Thomas

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Title: Sputum: Preparation and Examination Of Gram Stained Smears by Hilaire Thomas


1
Sputum Preparation and Examination Of Gram
Stained Smears by Hilaire Thomas
2
  • 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.

3
  • 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.

4
  • Equipment Required
  •  

5
  • 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 patients name,
    the date of collection, and the specimen
    accession number.
  •  

6
  • 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.

7
  • The specimen that is uniformly green and purulent
    presents no problem. Any portion of it will
    provide useful information.

8
  • 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.

9
  • Specimens may be heavily stained with blood.
    Select the portion of the sample that is mucoid
    and blood stained.

10
  • When only a few blood stained flecks are present,
    these are the portions to select.

11
  • Avoid any part of the specimen that is watery, as
    this is probably saliva.

12
  • Select purulent, mucoid, or blood stained samples
    for examination.

13
  • 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.

14
  • 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.
  •  

15
  • The sample should be about the size of a pea and
    should be placed near the center of the slide.

16
  • Orient the slides so that the frosted sides are
    facing each other.

17
  • 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.

18
  • Move the slides against each other in several
    directions with a rotating motion. This will
    spread the specimen between the slides.

19
  • To separate the slides, maintain the contact
    between the slides vertically and pull the slides
    away from each other horizontally.

20
  • Smears of Specimen on Two Slides

21
  • 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.

22
  • 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.

23
  • 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.

24
  • 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.

25
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.

26
Elements Present in Sputum
  • Cellular elements
  • Non-cellular elements

27
Cellular Elements of Sputum
  • Squamous epithelial cells
  • Respiratory epithelial cells
  • Polymorphonuclear leukocytes
  • Mononuclear cells
  • Alveolar macrophages

28
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29
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

30
Squamous epithelial cells are easily seen and
recognized under low power magnification.
31
The lining cells of the oropharynx, their
presence in a sputum smear usually indicates
contamination with saliva.
32
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33
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

34
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.

35
  • Respiratory Epithelial Cells

36
  • Under low power magnification, the cells are long
    and slender. They are sometimes seen in groups.
    They may appear spindle shaped.

37
  • Respiratory Epithelial Cells in Various Stages
    of Disintegration

38
  • 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.

39
  • 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.

40
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41
  • 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.

42
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

43
  • Alveolar Macrophages

44
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.
45
  • 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.

46
  • 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.

47
  • 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.

48
  • 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.

49
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50
  • 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.
  •  

51
Characteristics of Polymorphonuclar Leukocytes
  • Multi-lobed nuclei
  • Granular cytoplasm
  • Cytoplasm sometimes contains phagocytosed bacteria

52
  •  
  • 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.

53
  • At high power the intact poly is easily
    identified by its multi-lobed nucleus and
    granular cytoplasm.
  •  

54
  • The nuclei are often more difficult to see when
    the cells are imbedded in mucus.

55
  • 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.

56
  • 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.

57
  • 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.
  •  

58
  • 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.
  •  

59
Non-cellular Elements
  • Mucus threads
  • Curschmanns spirals
  • Bacteria
  • Yeasts

60
  • Mucus

61
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

62
  • Mucus is easily seen under low power
    magnification. It forms long irregularly shaped
    strands which may have cells and bacteria
    embedded in it.

63
  • Curschmann's Spirals

64
Curshmanns 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.
65
  • This is a portion of the same spiral under oil
    immersion magnification.

66
  • 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.

67
  • Streptococcus pneumoniae

68
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.
69
  • Staphylococcus aureus

70
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.
71
  • Haemophilus influenzae
  •  

72
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.
73
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.
74

Enterobacteriaceae These organisms are gram
negative rods but lack any more specific
differential characteristics. They are usually
short fat rods, larger than Hemophilus sp.
75
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.
76
  • 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.

77
  • Yeast

78
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.
79
  • Evaluation of Specimen

80
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.
81
The orange coloration of the alveolar macrophages
can be easily seen on this slide. Mucus, polys,
and other mononuclear cells are also present.
82
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.
83
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.
84
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.
85
  • 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.

86
  • This slide shows polys, alveolar macrophages and
    one contaminating squamous cell. This would be
    considered a good specimen, since contamination
    is minimal.

87
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.
88
  • The presence of respiratory epithelial cells is
    of little help in establishing the nature of the
    specimen. They are usually present in
    bronchoscopy specimens.

89
Reporting
90
  • Report the presence or absence of alveolar
    macrophages for all specimens.
  • Report either
  • Alveolar macrophages present,
  • or
  • No alveolar macrophages seen.

91
  • Report mucus only when it is present.
  • Report
  • Mucus present.

92
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.
93
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

94
  • 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"

95
  • 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.

96
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.
97
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.
98
  • 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.

99
  • You will recognize these as probable
    staphylococci.

100
  • 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.
  •  

101
  • Other Descriptive Terms
  • Specimen inadequate. Saliva only
  • Repeat requested
  • Squamous epithelial cells only
  •  
  • These terms are used together and may constitute
    the whole report.
  •  

102
  • Terms in previous slide are used when only
    squamous epithelial cells and mixed organisms are
    present as in this example.
  •  

103
  • 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.

104
  • 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.

105
  • Scanty specimen
  • This term is used when there is little cellular
    material to be examined. This type of specimen is
    usually inadequate for interpretation.

106
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.

107
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.
  •  

108
  • 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.
  •  

109
  • 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.
  •  

110
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