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. The equipment needed to prepare the smears consists of
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.

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
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
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
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
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

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.

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
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
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.

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
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
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.

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
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
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
Curschmann’s 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
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. Curshmann’s spirals are plugs of mucus which form in the bronchioles
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.

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
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
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
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.
“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
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
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
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.