Study of parasites

1. Cover slip and slide:
https://www.youtube.com/watch?v=FG74x15a0y8&ab_channel=JeromeReamer
Hanging Drop Method:
https://www.youtube.com/watch?v=O0DQ2LTARaU&ab_channel=AmritaVlab
First 2 minutes only:
https://www.youtube.com/watch?v=Z1yyuQ8qHQI&ab_channel=AmritaVlab
Alternative video to understand the movement of bacteria:
https://www.youtube.com/watch?v=ujzSmsmg7ok&ab_channel=SridharRao
Sewarage= Sewage
So, hanging drop method is the best method to watch live bacteria.
: Dry mount means placing sample directly on slide
without putting water. Wet mount means placing a sample in drop of water on
slide and then covering it with cover slip.
A dry mount is exactly as it sounds: the thing you plan to observe is
placed on the slide with a cover slip over it. No water is needed. This kind
of mount is usually used for inanimate objects or things that do not need
water to live.

2. Why is a wet mount better than a dry one?
The hanging drop and wet mount techniques allow for observation of living
organisms. The wet mount tends to dry out quickly under the heat of the
microscope light; it is simpler to perform, but it is useful for short-term
observation only.
Which organisms are used in the method?
Generally, Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus aureus,
Proteus.
HANGING DROP PREPARATION TO EXAMINE
THE MOTILITY OF MICROORGANISM / BACTERIA
Hanging Drop Preparation is the useful technique employed in the
laboratory for the microscopic examination of living microorganisms
(viable microorganisms), especially the bacteria without staining them
and to see their motility due to flagella.
PRINCIPLE OF HANGING DROP PREPARATION
The Hanging drop preparations is a special type of Wet mount in
which a drop of broth culture of microorganism or bacterial
suspension to be analyzed is placed on a glass cover slip which is
encircled with a stick substance, preferably the Petroleum jelly and
this cover slip containing the specimen is promptly inverted over the
special type of Glass slide known as Cavity slide or depression slide
containing a well (depression or cavity) in order that the drop hangs
freely on the cover slip in the concavity of slide and the Petroleum jelly
forms a seal that prevents the evaporation of the specimen and
preserves it temporarily.
The hanging drop preparation is then examined under the microscope
to check the motility of the organism, preferably under reduced light
to enhance the visibility and for better contrast. This method is ideally
used in the laboratory to check the motility of Bacteria.

3. REQUIREMENTS FOR HANGING DROP PREPARATION:
 Soil/ Sewage sample
 Normal saline (0.9 %)
 Hanging drop slide / Cavity Slide
 Glass Coverslips
 Vaseline / Petroleum Jelly
 Matchsticks / cotton swab (cotton swab, cotton bar, swab stick
Same)
 Inoculation loop
 Microscope
 Bunsen Burner
PROCEDURE:
⇒ Collect soil/ sewerage (sewage) sample and mix the sample with
normal saline.
⇒ Clean and flame the hanging drop slide/cavity slide and place it on
the table with concavity/depression side up.
⇒ Now, Clean a coverslip and apply petroleum jelly or vaseline on each
of the four corners of the coverslip, using a matchstick/ cotton swab.
⇒ Place the jelly coated coverslip on a clean paper with the petroleum
jelly side up.
⇒ Transfer one loopful of broth culture or Bacterial suspension in the
center of the coverslip.

4. ⇒ Now, Place the depression slide onto the coverslip, with the cavity
facing down so that the depression covers the suspension drop.
⇒ Press the slide gently to form a seal between the coverslip and the
slide to prevent the evaporation of specimen.
⇒ Lift the preparation and quickly turn the hanging drop preparation
coverslip up so that the culture drop is suspended in the concavity of
Depression slide.
⇒ Examine the preparation under low power objective lens, with
reduced light and close the diaphragm of the microscope. Focus the
edge (appeared as irregular lines crossing the field) of the slide using
the coarse adjustment knob.
⇒ Without moving the microscope tube, switch to high power
objective lens and examine the preparation again.

5. RESULT:
Motile bacteria are observed on the edge of the drop. The motile
bacteria were differentiated from bacteria establishing Brownian
motion.
OBSERVATIONS OF HANGING DROP PREPARATION
Observe under the microscope by focusing the edge of the drop and
carefully find the tiny objects which are bacteria. The bacterial cells
will appear as either a dark or slightly greenish tiny bodies, very small
rods (bacilli) or spheres (round or cocci).
Movements are seen under microscope.
PRECAUTIONS TO BE TAKEN WHILE PERFORMING
HANGING DROP PREPARATION
⇒ The use of PPE (Gloves, Mask, Lab coat / Gown, Safety goggles etc.)
is mandatory as you are going to deal with highly infections viable
microorganism.
⇒ Use the young culture of the organism as in the old cultures, most
probably the organisms are dead.
⇒ Put the appropriate size of the drop onto the cover glass which
should not be too large or too small and hang freely in the concavity of
depression slide.
⇒ First, observe under the low power objectives & then switch to high
power & oil immersion objective for easy findings.
⇒ Adjust the diaphragm accordingly for better contrast which
minimizes the errors in observations.
⇒ Observe carefully before reporting as Motile or Non- motile
organisms.
Discussion:

6.  Hanging drop method is an aseptic method for examining the
specimens from liquid culture instead of a solid culture medium.
 It is extensively used to study bacterial shape and
arrangement and presence of flagella.
 Specimens in the hanging drop method will show Brownian
movement, due to which the microscopic objects in the fluid will
swim erratically through the kinetic energy possessed by the
molecules in the surrounding fluid.
 The true mobility is observed through the multi-directional
movement of bacterial cells to longer distances, instead of the cells
moving back and forth. The bacterial motility can be observed
under 10X and 40X objective, which is shown in a diagram. In a
hanging drop method, 10X objective is initially used to focus the
microscopic image, then later the objective is raised to 40X to get
a magnified view of the sample taken and to distinguish between
the motile and immotile cells.
 The petroleum jelly used on the corner of the coverslip acts as
a sealing material between the coverslip and the concave
depression glass. Besides, it also reduces the evaporation and
excludes the effect of air currents.
 Overuse of petroleum jelly can give false results, as it may
squeeze towards the centre of the drop containing microorganisms
or may squeeze out of the edges and can stick to the objective
lens of the microscope.
 The morphology of spiral bacteria can be explicitly studied in
hanging drop method, as its shape becomes distorted in the heat
fixing method.
 Bacterial motility or mobility can be well studied by employing a
hanging drop method, in which a bacterial cell can freely move in
the liquid medium.
 It is a wet mount technique as the microbes were first mixed with
normal saline.
 The cytological changes that occur during the cell division, spore
formation and germination of the bacteria are the events that need
to be studied in a living condition or hanging drop method.
The cytoplasmic inclusions, including vacuoles, granules etc. are
easily noticeable by using this method.

7.  We should be careful because, It is risky for the study of
pathogenic bacteria in a living condition.
 And the depression slide is cost-effective, and the coverslip is
fragile to work with.