Practical laboratory 3

Gram staining and quality control. Bacterial identification. Stainings used in Bacteriology
and Parasitology
(coproparasitologic exam, anal imprint and recognition of the parasites in the blood)

Objectives:
1. To be able to identify the parts of the microscope and to correctly work with it, to
performe a microscopic examination, as first step of bacteria identification.
2. To know the steps in performing a smear in the microbiology laboratory.
3. To know the types of solution used in microbiology laboratory.
4. To know the steps of Gram staining and to interpret the results of quality control for this
staining.
5. To know the importance of Gram staining, for identification of bacteria based on
microscopic characters and targeting of antibiotic therapy prior to finding out suscep-
tibility spectrum (empirical treatment)
6. To correctly describe the morphotynctorial features of the bacteria observed in the
microscopic exam of a stained smear.
7. To know the advantage and disadvantage of microscopic exam.
8. To know the specifics of coproparasitologic exam.
9. To know the indications of anal imprint.
10. To know the peculiarities of parasitologic blood exam.

1. The optical microscope contains:

- mechanical part (focus knobs - to move the stage), coarse adjustment, fine adjustment, stage
to hold the specimen);
- optical part (lenses system: objectives, occulars);
- light system.
The power or magnification of a compound optical microscope is the product of the powers
of the ocular (eyepiece) and the objective lens.
The maximum normal magnifications of the ocular and objective are 10× and 100×
respectively, giving a final magnification of 1,000×.

2. Microscopic preparations

2. a. wet microscopic preparation

Indications:
- highlighting the mobility of microorganisms;
- observation of fungi, protozoa, spirochetes;
• dark field microscopy for spirochetes
- detecting and fast identification of some cellulars elements in pathologic products.

2.b. Stained microscopic preparation

Indication: observation of the morphological features of bacteria (shape, arrangement, some
special strctures) and Gram afinity.

1
The smear: the microbial material (pathologic product, bacterial culture) displayed in a thin and
uniform layer on the surface of microscope slide.

The steps of preparing the smear:

1. Display
2. Dry
3. Heat setting.
The types of staining:
- simple: blue methylen: show the morphology of bacteria
- differential: Gram
Ziehl Neelsen
- special staining (flagellum, spores)

3. Gram staining
Principle: see the lectures, regarding the bacterial cell wall structure

The steps of Gram staining:

Nr. Step Describtion of the step Gram Gram
crt. positive negative
bacteria bacteria
1 Staining Crystal violet, 1 minute, wash step Violet Violet
2 Fixation Lugol, 2 minutes, no washing Violet Violet
3 Differentiation Alcohol – acetone mix, 5 – 10 seconds, wash Violet Colourless
4 Counterstaining Ziehl solution dilluted 1/10, 30 seconds, Violet Red
wash

Rezultat: Gram positive bacteria: violet

Gram negative bacteria: red
Quality control:
- control Gram smear: suspense which contains a mix of gram positive and gram negative
bacteria (e.g., gram positive cocci and gram negative bacilli, to be also different shapes) (Fig. 1).
- on the smears from pathologic products, the leukocytes are all the time red.
• Red leukocytes and gram positive bacteria: a good Gram staining procedure
• Red leukocytes and gram negative bacteria: we should examen the control Gram
smear.
Sources of errors: -under - decolorisation
- super - decolorisation

Fig. 1: Quality control

2
 4. The medical importance of Gram staining:
• It is the first step in bacterial identification
• Guidance of antimicrobial therapy for first intention, according with their Gram
affinity and shape
• Guidance in choosing the appropriate culture media useful for the next steps of
identification

5. Oil immersion microscopy and description of a smear:

Examination :
- calculation of the magnification microscope
- the correct technique description
Description:
-shape – cocci (Fig. 2; Fig. 3; Fig. 6; Fig. 7)
- bacilli (Fig. 4; Fig. 5)
-shape variation
- round cocci (staphylococci, streptococci), ovalar (streptococci), lancet shape
(pneumococci- Fig. 6), coffee beans shape (Neisseria- Fig. 7);
- bacilli with round / cut ends (Bacillus Genus), end clubs (diphtheric bacilli)
-arrangement: in clusters (staphylococci- Fig. 2), in chains (streptococci- Fig. 3), in diplo
(pneumococci), chinese letters (diphtheric bacilli)
-relative dimension: small, big
-gram afinity: gram positve / negative
-presence of capsule: pneumococci
- presence of spore:
- shape: ovalar (Clostridium/Clostridioides Genus), sferical (C. tetani; Bacillus Genus)
- arrangement: central (Bacillus Genus), terminal (Clostridium tetani- Fig. 5),
subterminal, paracentral, etc (Clostridium/Clostridioides Genus)
- dimenssion: big, small

The advantages of microscopy:

- rapid method to microorganisms detection;
- low cost and easy to perform;
- is guiding to the next microbiological diagnosis methods;
- can highlight non-cultivable bacteria or those with slow growing;
- is offering quantitative information (1 bacteria/microscopic field~ 105 CFU/ ml). These
information are useful in interpretation of the results (e.g., the microscopic exam in infections
with conditionate pathogenic microbes), which can belong to normal flora, but also, in some
conditions, can be etiologic agents.

The disadvantages of microscopy:

- low sensitivity and low specificity.
Sensitivity is refering at the positivity rate – a culture or a pathologic product with ˂ 103
CFU/ml will generate a false negative result (microscopy can highlight bacteria over 103
CFU/ml).
The importance in diagnosis: where there is a suggestive clinico-epidemiologic context, but
with negative microscopy, next step is followed by the culture (cultivation being more
sensitive).
Specificity: the microscopic features are seldom enough to define the species (S. penumoniae,
Corynebacterium diphteriae, C. tetani), being only indicative, guiding to the conclusion:
bacterial genus (Staphylococcus, Streptococcus (Fig. 3), Bacillus, Clostridium) or just to
bacterial families (Enterobacteriaceae (Fig. 4), Pseudomonadaceae etc.).
3
Fig. 2.: Staphylococcus Fig 3: Streptococcus

Fig 4: Gram negative rods (bacilli) Fig. 5: Clostridium tetani

Fig. 6.: inflamatory cells (PMN), and Fig. 7.: PMN, and Neisseria
Streptococcus pneumoniae

4
The coproparasitologic exam:
- The pasitologic exam of feces – is the diagnosis method for detection of parasites
(protozoa, helmints) with intestinal localisation or which are eliminated by digestive tube.
- The microscopic exam:
▪ Direct – wet smear – in physiologic serum or in Lugol solution – detection of protozoa
(trofosoids and chists, ex. Giardia), helmints eggs (ex. Ascaris), blood, mucus. The study
of mobility and of morphology.
▪ Smear in thick layer (Kato-Miura method) – detect and identify the eggs of helmints with
intestinal localisation (ex. Ascaris)
▪ Smear after concentration of parasitary elements – for aliquots with smaller number of eggs
a) Concentration by flotation (hipertone solutions) – for easy eggs (ex. Taenia)
b) Concentration by sedimentation (hipoton solutions) – for heavy eggs (ex. Ascaris)
▪ Stained smear – identification of protozoa (trofozoids and chists), helmints eggs
- May-Grunwald-Giemsa, Ziehl-Neelsen stainings

Anal imprint – diagnosis of pinworm and tenia with Taenia saginata

- sampling from anal creases with a adesive band (scotch) and a glass rod

The parasitologic exam of blood

- Direct exam - smear stained with May-Grunwald-Giemsa – allow highligthing of
parasitar elements (species diagnosis) and red blood cell modification
- The heavy drop – concentration method – difficult diagnosis of species (the parasites
appear shrink)