LAMINAR AIR FLOW
Principle
: The laminar air flow is based
on the flow of air current of uniform velocity along parallel flow lines, which
help in transferring microbial cultures in aseptic condition. Air is passed
through HEP A (High Efficiency Particulate Filters).
Construction
and working
rl.aminar air is an apparatus consists of an air blower in the rear side of the
chamber which can produce air flow with uniform velocity along parallel flow
lines. There is a specific filter system of high efficiency particulate air
filter (HEP A) which can remove particle as small as 0.3 mm.
Infront of the blower there lies a mechanism through
which air blown from the blower produces air velocity along parallel lines.
The laminar flow current is
based on flow of air current of uniform velocity along parallel flow lines
which help in aseptic conditions. Air is passed through the filters does not
allow any kind of microbe to enter into the system. Inside the chamber one
fluroscent tube and other UV tube are fitted.
Two switch for these tubes and
a separate switch for regulation of air the air flow are fitted out side the
apparatus.
Initially dust particles are
removed from the surface of the laminar flow with the help of smooth cloth
containing alcohol. Switch on the UV light for a period of 30 minutes so as to
kill the germs, if any present in the area of working space.
The front cover sheet of the
apparatus is opened to keep the desired material inside. The air blower is set
at the desired degree so that the air inside the chamber is expelled because
the air inside the chamber may be contaminated.
Applications: It is used to reduce the danger
of infection while working with pathogenic micro-organisms and to prevent
contamination of sterile materials.
COLORIMETER
Principle: Colorimeter IS the instrument used
for the measurement of coloured substances. The instrument is operative in the
visible range (400 - 800 nm) of the electromagnetic spectrum of light. The
working of the colorimeter is based on the principle of Beer - Lambert's law.
Beer -
Lambert law states that the amount of transmitted light decreases exponentially
with an increase in the concentration of absorbing material (i.e. the amount of
light absorbed depends on the concentration of the absorbing molecules) and
according to lambert's law, the transmitted light decreases exponentially with
increase in the thickness of the absorbing molecules (i. e. the amount of light
absorbed is dependent on the thickness of the medium).By combining the two laws ( Beer - Lambert law) the
following mathematical derivation can be obtained.
I = IOECI
Where I = intensity of the transmitted
light. 10 = intensity of the incident light.
E =
molar extinction coefficient ( characteristic of the substance being
investigated)
c =
concentration ofthe absorbing substance (moles/ I / or g/dl)
t =
thickness
of medium through which light passes
when
the thickness of the absorbing medium is kept constant (i.e. lamberts' law),
the
intensity of the transmitted light depends only on concentration of absorbing
material. In other words, the Beers' law is operative.
If
the ratio of transmitted light (I) to that of incident light (l)
is
reffered to as
transmittance
(T).
T =
II 10
Absorbance
(A) or optical density (OD) is very commonly used in laboratories. The relation
between absorbance & transmittance is
expressed by the following equation.
A =
2
-logl0 T= 2 -log T
Construction: The colorimeter consists of
light source, filter sample holder and detector with display (meter or
digital). A filament lamp usually serves as light source. The filters allow the
passage of a small range of wavelength as incident light. The sample holder is
a special glass cuvette with a fixed thickness. The photoelectric selenium
cells are the most common detectors used in colorimeter. The diagrammatic
representation of colorimeter is Light source - filter - cuvette - sample hoder
- Detector - display
NEUBAUR'S
HAEMOCYTOMETER
Principle:
The
ruled area of the hemocytometer consists of several, large, 1 x 1 mm (1
mm 2) squares. These are subdivided
in 3 ways; 0.25 x 0.25 mm (0.0625 mm 2), 0.25 x 0.20 mm (0.05 mm 2
) and
0.20 x 0.20 mm (0.04 mm 2 ). The central, 0.20 x 0.20 mm
marked, I x 1 mm square is further subdivided into 0.05 x 0.05 mm
(0.0025 mm 2 ) squares. The raised edges of the
hemocytometer hold the coverslip 0.1 mm off the
marked
grid. This gives each square a defined volume.
Construction:
The Neubaur's haemocytometer slide has 2 raised platform separated by a horizontal
groove. This groove joins 2 vertical grooves so that they form 'H' shaped area.
There is a raised platform on either side of central groove. The outer
platforms are at hidher level than the central platform, the height between the
central platform and lower surface of the groove is III 0 of a mm. each central
platform has a counting chamber. So that 2 preparations can be set at a time.
Each counting chamber is a square of 3mm. so that it encloses the arera of 9
sq.mm. Each counting chamber is divided into 9 squares. Each having an area of 1 square mm. each of corner
counting chamber is divided 16 squares. These are used for counting WBC. Each
small square has an area of 1116 of a square mm and the volume of 1 II 0 of the cubic mm. the coverslip usually used
is slightly bigger than the ordinary coverslip.
The
central square marked R is used for counting RBC this is divided into 25
small squares by triple lines. Each small square of
RBC counting chamber is divided into 16 smallest squares by single lines. Each
smaller square of the RBC counting chamber has an area of 1/400 sq.mm and a volume of 1/4000 cubic millimeter.
The
WBC pipette is a capillary tube open at both the ends with a central bulb.
One
end of the tube is narrow while the broad end. The bulb contains a white glass
bead. There are 3 graduations marked namely 0.5, 1 and 11 is above the bulb.
The RBC pipette is a capillary
tube open at both the ends. It has a central bulb containing a red glass bead.
One end of the tube is narrow while the other end is broad. A plastic tube is
attached to the broader end of the tube. There are 3 marks 0.5, 1 and 101. Of
these 0.5 & 1 are found below the bulb and
101 found above the bulb.
Applications:
The
neubaur's haemocytometer is used to count the number of RBC and WBC cells and
is also used to count the fungal spores in liquid suspension.
.
.
CENTRIFUGE
Principle: This method is based on the
principle of sedimentation i.e. rotation of substance around the central axis
at high speed and separates substancesl particles on the basis of mass and
density by means of centrifugal force. The centrifugal force is noted in
revolutions per minute (rpm) of angular speed.
Construction and working: A centrifuge consists of a
"head" which is rapidly revolved by an upright motor. Generally four
metal cups or containers are attached to the head holding tubes or other
containers of the material from which the particulate matter is to be
separated. During centrifugation, liquid containing particulate matter is kept
in the tubes, runs at a particular speed and when centrifugation is complete,
the particulate matter gets settled at the bottom of the tubes.
Applications: centrifuge is used to separate
particles dispersed in a suspended matter, separation of mixtures of liquids
varying in their density or solids from liquids or concentrating microorganisms
in various samples in enzymatic studies.
INOCULATING LOOPS
PREPARATION OF POTATO DEXTROSE AGAR MEDIA
QUEBEC COLONY COUNTER
It is a device used to count the number of colonies in
a plate. In this a Petri dish having colonies of microorganisms developed on a
solid medium is mounted on a platform. When the Petri dish is illuminated from
beneath, the visible colonies can be counted by a lens of XI.S magnification. Each
colony touched by the electrode is recorded automatically in the counter.
INOCULATING LOOPS
Are the most commonly used
tools for aseptic transfer. An inoculating loop consists of an insulated handle
provided with srew device at the top which holds a heat resistance nichrome or
platinum wire, approximately three inches long. The wire end is bent round to
form
a loop.
PREPARATION OF POTATO DEXTROSE AGAR MEDIA
Aim:
To
prepare potato dextrose agar medium.
Principle: In nature,
microorganisms grow on a natural media or the nutrients available in water,
soil and dead or living organic material. In a laboratory they are grown in
synthetic media. Cultivation of microbes in the lab requires an artificial
medium. The food base that supports the growth of an organism is called culture
medium. The microorganisms (i.e. bacteria, fungi, yeast & protozoa) and plant and animal cells can be
grown in pure cultures for experiments. For industrial use, the culture media
are devised in such a way that the organisms should get all the nutritional
requirements I.e. carbon source, nitrogen source,trace elements, hormones,
vitamins, aminoacids, nucleotides etc.
Laboratory media are generally
synthetic media prepared from maerials of precise or reasonable well - defined
composition. Such media contain peptone as a common ingredient which provides
small peptides that microorganisms can use other substances such as yeast
extract, beef extract, casein hydrosylate (from milk), serum, blood, or heated
whole blood are also used in most routine laboratory cultures.
The
diagnostic media are :
1, Selective media , if they encourage the growth of
some organisms and inhibit the growth of others,
2.
Differential media, if they allow different kinds of
colonies on the same plate to be distinguished from one another .
3.
Enrichment media - if they provide a nutrient that
encourages growth of a particular .
microorganism.
Agar is used as solidifying
agent, has no nutritive value and such media with agar are called as nutrient
agar media. When agar is not added, medium remains in liquid form, is called as
nutrient broth.
Requirements:
·
Peeled potato tuber (200g), Dextrose (20g), Agar
(20g), Beakers, Conical flasks, Potato peeler, Knife, Muslin cloth, Sterilized
water, Measuring cylinder, Non absorbent cotton, Autoclave, Hot air oven, News
papers.
Procedure:
l.
Take 500 ml of water in a beaker.
2.
Add 200 g washed, peeled and sliced potatoes to the
beaker.
3.
Boil potatoes gently for 30 minutes or the time till
they are easily penetrated by glass
rod.
4.
Filter through muslin cloth, squeezing out all liquid.
5.
Add 20 g dextrose to the potato extract.
6.
Take 500 ml of water in another beaker and heat it.
7.
Add 20 g agar, bit by bit to the hot water to
disslolve it.
8.
Mix agar with the potato extract.
9.
Bring volume up to 100 ml by the addition of distilled
water.
10. Transfer the medium
into the conical flasks. Plug the flasks containing medium.
11. Transfer all the
glass wares, conical flask with media into the metallic basket of autoclave.
12. Sterilize at 121°C,
15lbs pressure for 30 minutes in an autoclave.
13. Petridishes and
other glass wares required can be sterilized by hot air oven (180° C for
one hour). The glass wares are wrapped with newspapers
before placing them in oven.