1. EVANGELICAL CHURCH IN
ZAMBIA
MUKINGE SCHOOL OF NURSING
MICROBIOLOGY
MCB 018
HAANTOBOLO A.
BSC. BIOMED. MPH ( C)

2. STAINING
• This is an artificial colouring of tissues OR cells to
facilitate the identification and examination under
the microscope.
• The microscopic examination of stained properties
enables the morphology, relative size and
arrangement of micro-organisms to be seen clearly.
• STAIN
• Stains are chemically salts which can be base, acid,
or neutral depending on whether the colouring
component is contained in the base, acid or both
parts of the stains.
• Most stains used to stain bacteria are of basic types
such as crystal violet, basic fuchsine and methelen
blue.

3. • Mordant
• The chemical which helps to bring about the stain
reaction e.g. iodine in Gram stain and phenol in
Ziehl Nelson (ZN)
• Helps the cells to absorb the primary stain
• Decolourization
• This is the selective removal or washing out the
excess stain.
• Counter Stain
• The stain is taken up by the organism which has
been decolourised to provide a good background.

4. MAKING OF SMEARS/PREPARATION
OF FILMS
• Use clean slide free from greasy
• Mark with a glass writing diamond pencil
• Make the smear from liquid, solid media or swab
spread out smear about 3X2 on the microscope
slide centre air dry the smear.
• Label the slide with patient name, ID number and
date
• Fix by passing it 3 times over a flame (if
appropriate) cool and stain.

5. 2.2 MICROSCOPIC EXAMINATION
• Microscopy is the technic of using microscopes to
view samples and objects that cannot be seen
with the unaided eye (objects that are not within
the resolution range of the normal eye).
• A microscope is an instrument used to see objects
that are too small for the naked eye.
• Types of Microscopes
• i. Light Microscope
• ii. Electron microscope (mostly for viruses)
• iii. Flourescent microscope

6. Electron microscope

7. Light Microscope

9. STAINING METHOD
• LEOFFLER’S ALKALINE METHYLENE BLUE
• This is a basic dye for routine use in studying the
morphology of micro – organisms in smears from
cultures.
• The stain is more intense than natural solutions
of methylene blue and may
• show some degree of poly chromatic staining
(many colours) chromatic means a change in
colours.

10. METHOD
• The heat fixed smear/the slide is flooded
(applied) with methylene blue stain which is
allowed to act for 3 minutes, 30 seconds
• Then wash off with water and the slide
blotted dry
• Examine under oil immersion.
• Bacteria stain deep blue
• Pus cells stain deep blue

11. GRAM’S STAIN
• Gram’s stain shows up bacteria and also divides
them into 2 groups, gram positive and gram
negative. Depending on whether they can be
decolourised or not
• The film is first stained with methyl violet, or
crystal violet and treatment with iodine (mordant)
which color the bacteria a deep violet colour.
• The film is then washed with acetone and which
decolorizes the gram negative bacteria.
• Those which retain the primary colour after
washing are known as gram positive and those
which are decolourized and take up a red counter
stain are called gram negative.

12. SOLUTIONS REQUIRED FOR GRAM STAIN
• 0.5% Crystal violet
• Lugols iodine
• A cetone / alcohol
• Neutral red
• METHOD
• Prepare a smear, allow to dry fix with gentle heat.

13. • Slide is flooded with solution 1 (crystal violet)
which is allowed to act for about 3 minutes.
• The slide is washed with water and Iodine’s
solution is added and let to stand for 3minutes.
• Rinse with water.
• The slide is decolourised with gram’s
defferentiator (Acetone) for only about 15
seconds.
• Wash with water.
• Apply solution (neutral red) and allow to act for
2 minutes.
• Rinse with water and blot dry slide.
• Examine under oil immersion.

14. RESULTS
• Gram positive organisms stain violet
• Gram negative organisms stain red

15. ZIEL – NEELSEN’S STAIN (ACID – FAST STAIN)
• This is used for the identification of tubercle bacilli
(mycobacterium tuberculosis and Mycobacterium
leprae ).
• Which do not stain with ordinary dyes due to a very
resistant outer envelope of a fatty nature which
prevents penetration of the stain but can be stained
with
• The carbol fuchsin – a bright red stain colours all
bacteria.
• The tubercle bacillus differs from other bacteria in
being difficult to stain and long resistance to
decolourisation once the red stain has penetrated.
• Because of its resistance to decolourisation by acid-
alcohol it is known as Acid –Fast Bacilli (AFB) or Acid-
Alcohol Fast bacteria (AAFB)

16. SOLUTION
• Carbol fuchsin 20%
• 3% acid alcohol/sulphuric acid
• Malachise green /methylene blue

17. METHOD
• Prepart 2-3 cm smear, allow to air dry and fix by
flaming (pass it 3 times in flame for smear to stick
on slide)
• Flood slide with carbol fuchsin and then heat
under neath the slide until steam rises, leave to
stain for 5 minutes
• Rinse the slide in running water
• Apply 3% acid alcohol f 3 minutes
• Wash with water the
• Apply malachite green/methylene blue for 5
minutes (counterstain)
• Wash slide with running water blot and air dry.
• Examine under oil immersion.

18. RESULTS
• Acid alcohol fast bacteria appear bright red
• Background and other bacteria and pus cells
appear green/blue

19. OTHER DIFFERENTIAL STAIN
• WRIGHT’S STAIN: This is a mixture of eosin and
blue it is used to identify blood cells and malaria
parasites
• Giemsa’s stain: A solution of eosin, glycerine and
methanol it is used to identify trypanosomes,
Malaria parasites and rickettsia.
CONCLUSION
• The optimal understanding of microbiology by a
nurse is paramount to effectively provide
comprehensive, holistic care to patients.
• Microbiology provide knowledge about
microorganisms and how they cause disease.