Culture, Staining, Requirements and Observations
Essentially, sputum is composed of a mixture of
saliva and mucus. Also referred to as phlegm, sputum is produced in the bronchi
and bronchioles of the respiratory system and coughed up from the lower
airways and should not be confused or mistaken for saliva, which is
secreted in the mouth for digestion purposes.
It should also not be confused
with mucus given that sputum is only produced in parts of the respiratory tract
(such as bronchioles). It may vary in terms of color and viscosity ranging
from thick to thin or clear to dark yellow in color. All this largely depends
on its contents. For instance, blood cells or tar may be present as well
as bacteria. Sputum microscopy is used to determine
any underlying health issues for treatment.
Refers to the microscopic
investigation of sputum. This has been shown to be one of the most efficient
methods of identifying tuberculosis infection among patients in order to help
them start treatment.
For microscopy, sputum may be prepared directly
once collected or cultured before microscopy.
Culture simply involves encouraging the microorganism in the sputum to grow. The sputum may contain given types of bacteria or fungi.
Using the culture method, these microorganisms are able to grow in a conducive environment (with sufficient nutrition, optimal temperature and moisture etc) making it easier to identify them.
For sputum culture, it is advised not to use mouthwash among other medicinal substances used for mouth washing. This is due to the fact that they may contain various antibacterial substances that would have a negative impact on the final results.
The sample is either obtained by inducing or being coughed up (expectorated) early in the morning before drinking or eating anything.
Depending on the type of microorganism suspected
to be present, the following are some of the growth media used in sputum
- Blood agar
- Chocolate agar
Selective media for:
- Pseudomonas species
- Fungi and yeasts
- B. cepacia complex
* Once the microorganism starts to grow, it is
identified using a microscope.
With regards to microscopy, bright field and
fluorescence microscopes can be used for observing and studying the appearance
of acid-fact bacilli. On the other hand, gram stain method can be used for the
purposes of detecting the presence of fungi.
A bright field microscope is the most basic form
of microscope illumination techniques with regards to compound microscopes. For
this technique, no special additional accessories are required in a compound
microscope, which means that the user will view the specimen as a darker object
that is surrounded by a bright field (background).
Ziehl-Neelsen Technique (Acid Fast Bacilli Staining)
Ziehl-Neelsen staining method is an acid fast
staining method used to determine the presence of acid fact bacteria such as
the Mycobacterium species. The bright field microscopy is used for this
To determine whether a patient has TB, sputum microscopy is used to
detect the presence of mycobacterium tuberculosis, which is the bacteria that
- Bright field compound
- Staining rack
- Burning stick
- Slide rack
- Water/distilled water
- 25 percent sulphuric acid
- Carbol Fuchsin
- Methylene blue/Malachite
- Microscope glass
*Make sure to use a pair of gloves when handling
biological fluids like sputum.
- Carefully open the
container (that contains the sputum sample) and using a laboratory burning
stick (dry) obtain and spread a small amount at the central part of a
microscope glass slide - Use rotational movement to create a good smear
- Place the slide on a drying
rack and allow to dry for about 30 minutes or use a dryer to dry the smear
- Pass the slide over the Bunsen
burner flame 3 to 4 times to heat fix while avoiding to overheat
- Place the slide on the
staining rack and pour the Carbol fuschin stain (to cover the smear) and heat
until it starts evaporating - do not overheat
- Allow the slide to stand
for between 4 and 7 minutes then wash wish water
- Pour 20 percent sulphulic
acid on the smear and allow it to stand for a minute. Repeat this until the
smear appears pink in color
- Wash the slide with water
and cover the slide with malachite green stain or methylene blue and allow the
slide to stand for about 2 minutes
- Wash the slide with water
and allow the slide to dry on the drying/draining rack - use a tissue paper to
clear the slides and back of the slide
- Observe the slide under the
microscope using 100x oil immersion objective
If the bacteria is present in the sample,
students will see pink rods that are either straight or slightly curved while
the background will appear bluish in color - the pink rods are the bacteria.
Acid fast bacteria like mycobacterium have a
lipoid capsule that gives their cell walls a waxy appearance. Their cell walls
also contain large amounts of mycolic acids and fatty acids. Due to these
compounds in their cell wall, acid fast bacteria require a special technique
with regards to staining.
During staining, the primary stain is able to
penetrate and enter the cell wall of acid fast bacteria because the stain is
lipid soluble. Heating also enhances this. When a decolorizer is used
(sulphulic acid) acid fast bacteria retain the primary stain while the non-acid
fast microorganism loses the stain.
Acid fast bacteria are able to retain the
primary stain because of the characteristic of their cell wall. When the
counter stain is used, it is readily taken up by the non-acid fast microorganisms,
but not the acid fast bacteria.
A fluorescence microscope is a compound
microscope that applies the use of fluorescence and phosphorescence to observe
the object. With this microscopy technique, the specimen under investigation is
the source of light. This technique is also important for sputum microscopy.
Also referred to as Truant staining method,
Auramine-Rhodamine staining method is one of the methods used for viewing and
studying the Acid-fast bacteria (bacilli). This method is not only easy, but
also quick to use, which has made it one of the best alternatives to
Ziehl-Neelsen technique today.
- Auramine Rhodamine Stain
- 0.5% Acid alcohol
- 0.5% Potassium Permanganate
- Microscope glass slide
- Fluorescence microscope
- Staining rack
- Drying rack
- Bunsen burner
Using a burning stick (or wire loop) obtain and
make a small smear at the center of a glass slide
Pass the slide over the Bunsen burner flame 3 to
4 times to heat fix
Flood the slide with Auramine stain and allow to
stand for 15 minute - cover the smear with the stain
Rinse the smear with distilled water until no
color remains - do not use chlorine water
Flood the slide with Acid alcohol for about 3
Wash the slide using distilled water
Flood the slide with potassium permanganate for
about 2 minutes
Rinse with distilled water and allow to dry
Observe under the microscope starting with low
magnification and then high magnification
If Acid fast bacilli are present in the sample, students
will see them appear as yellow or bright orange in color with a dark
Auramine-Rhodamine (primary stain) is a
fluorochrome dye that has affinity for acid fast organisms. When it comes in
contact with the cell wall of the bacteria, it forms a complex with the mycolic
acid present in the cell wall. Heat fixing enhances this process making it
difficult to decolorize when acid alcohol is used. Here, the secondary
stain/counter stain used is potassium permanganate.
This stain is used to make
other debris non-fluorescent so that they may not be seen when viewing under the
microscope. Therefore, only the cells that take up the primary stain can be
Gram staining method is used for the purposes of
distinguishing between gram positive and negative bacteria. This technique has
also been use for studying sputum.
- Sample (sputum)
- Glass slide
- Bunsen burner
- Compound microscope (bright
- Burning stick/cotton swab
- Using a burning stick or
cotton swab, obtain a small amount of the sample and make a smear at the center
of the glass slide - try making a thin slide
- Place the slide on drying
rack and allow to dry
- Pass the slide over the
flame several times, but avoid overheating. Simply pass it over the flame
several times - heat fix
- Flood the smear with
crystal violet and allow to stand for about a minute
- Tilt the slide and rinse
with distilled water
- Flood the slide with Gram's
iodine for about one minute
- Tilt slide and rinse with
- Tilt the slide and apply
the alcohol drop by drop until it runs clear (95 percent ethyl alcohol/acetone)
- Rinse with water
- Flood the slide with
safranin for about a minute
- Tilt slide and rinse with
- Blot the slide dry
When viewed under the microscope, students may
observe different types of cells (Squamous epithelial, mononuclear cells,
respiratory epithelial cells etc) and other cells like Streptococcus pneumoniae, staphylococci and Hemophilus influenzae if they are present. While the body cells may
appear pinkish, the microorganisms will appear darker having retained the
primary color due to their thick peptidogycan layer on their cell wall.
*By observing and studying the types of cells
and microorganisms present in the smear using gram-staining, it is possible to
tell the type of infection that the patient has.
Related: Blood Smear, Urinalysis
Return to Microscopy Applications
Return from Sputum Microscopy to MicroscopeMaster Research Home
Dr. Annette Jepson (2008) Laboratory Techniques
for the Diagnosis of TB – what’s available?
D. J. Flournoy (1999) Interpreting the Sputum
Gram Stain Report.