Gram Stain Worksheet


The Gram stain procedure was originally developed by the Danish pharmacist Hans Christian Gram to
differentiate pneumococci from Klebsiella pneumonia. The Gram stain is particularly useful in the
presumptive diagnosis of bacterial meningitis, bacterial pneumonia, bacteriuria, gonorrhoea, and
pyogenic infections of the brain, lung, abdomen, pelvis, and wounds.

Statistically about one third of all patients admitted to general hospitals have, or develop,
infections, a large proportion of which are caused by bacteria and fungi. The investment of time
spent in preparing and examining a Gram-stained smear when a specimen is first obtained often
yields valuable results that cannot be obtained from culture of the specimen alone. The
Gram-stained smear is also useful in judging the adequacy of the specimen obtained. In sputum and
urine specimens, for example, a poorly collected or contaminated specimen can be recognised by the
presence of many epithelial cells in the smear.

Information obtained from the Gram-stained preparation rarely permits definitive identification of
organisms, but usually narrows the possibilities in diseases such as gas gangrene, pneumonia or
meningitis, which have a variety of causative agents. Early diagnostic information obtained from
Gram-stained smears often allows the physician to prescribe narrow-spectrum antimicrobial therapy,
thereby reducing the risk of toxicity, superinfection, and the expense of broad-spectrum
"poly-pharmacy".

Moreover, in certain infections, such as Vincent's angina (associated with fusobacteria and
spirochetes), the organisms are not identifiable by the culture techniques employed in most
diagnostic microbiology laboratories, and the Gram-stained smear together with the clinical
findings form the basis for diagnosis.


Decide if these statements are true or false.

1 H. C. Gram developed the staining procedure because he had problems with pneumonia.

2 More than half of the patients in hospitals get an infection of some type.

3 Epithelial cells in the smear mean that the sample is of bad quality.

4 Thanks to the data from Gram-stain smear, we can identify a disease precisely.

5 In certain infections, the Gram-stain smear is the only basis for diagnosis.


Preparing a smear - video

Listening

A What are the steps to follow?

1 rinse with water

2 rinse with water

3 rinse with water

4 immediately rinse with water

5 dry the slide

6 decolourise with alcohol

7 put the Gram’s iodine on (for 10 sec)

8 put safranin on (for 30 sec)

9 fix the smear

10 flood with crystal violet (for 30 sec)


B Answer these questions:

1 Why is Gram-stain called differential?

2 How long does it take to have a result?

3 What type of bacteria are resistant to penicillin?

4 Which of the stains is called mordant? What happens if you forget to use it?

5 For how long do you put alcohol on the stain?

6 What kind of the counter-stain is used here?

7 What do you do before you put the stain under the microscope?


Gram Stain : Use of English

The first consideration is the correct ________ (prepare) of the smear. Make a thin film of the
material on a clean glass slide, using a sterile loop or swab. Air-dry, then heat-fix the slide by
passing it several times through a flame. Failure to follow these _________ (direct) may cause
staining artefacts and break the normal morphology of bacteria and cells.

The Gram staining ________ (proceed) itself involves the application of a solution of iodine
(potassium iodide) to cells previously stained with crystal violet or gentian violet. The cells are
next treated with a __________ (decolourise) agent such as 95 % ethanol or a mixture of acetone and
alcohol. While Gram-positive bacteria retain iodine-dye complexes after the treatment with the
decolourising agent, Gram-negative bacteria do not retain complexes when decolourised. To ________
(visual) decolourised Gram-negative bacteria, a red counterstain such as safranin is used after the
decolourisation treatment.


Grammar Point

Verb patterns