Use and handling of microscope, laminar flow, vacuum pumps, viscometer, thermometer, Preparation of media and methods of sterilization

1. Use and handling of microscope, laminar
flow, vacuum pumps, viscometer,
thermometer, Preparation of media and
methods of sterilization
PGS 504 :-Basic concepts in laboratory techniques
o.p parihar

2. Microscope
A microscope (from the Ancient Greek: mikrós, "small" and skopeîn, "to look"
or "see") is an instrument used to see objects that are too small for the naked eye.
The science of investigating small objects using such an instrument is called
microscopy.
Microscopic means invisible to the eye unless aided by a microscope.

3. Using of microscope
 Place the microscope on a stable place on a laboratory bench. Place a stool
behind the laboratory table for sitting to look into the microscope. The stool
should have a convenient height to let you see through eye piece without
bending.
 Place microscope near the window if daylight is used for illumination. Provide
illumination from any of the three sources - build in lamp, external lamp or
sunlight. Use the concern side of the mirror. Use the plane side of the mirror if
there is condenser.
 Direct the path of light to pass through the hole of the stage with maximum
intensity while setting the mirror.
 Put the slide between the clips provided on the stage.
 Revolve the nosepiece and align the low power objective (10X) to examine the
object on the slide.

4.  Adjust illumination to improve the clarity of the object on the slide.
 Put one hand on the focusing knob (coarse or fine) and another on the
screw to move the stage. The low power objective is commonly used to
screen the field of view. Bring the objective of interest in the centre.
 Switch to high power (40X) and increase illumination as needed. Repeat
the process of focusing.
 After screening under the low power and examining under high power, if
desired and required, oil – immersion objective is used for obtaining greater
details of the object.

5. Handling of microscope
 Microscopes are devices used to magnify small objects, allowing them to be seen
by the naked eye.
 Most microscopes have several different powerful lenses attached to them,
allowing the viewer to inspect the content at more than 100 times its actual size.
However, microscopes are extremely expensive, so you want to make sure you
handle the device properly.
 Hold the microscope with one hand around the arm of the device, and the other
hand under the base. This is the most secure way to hold and walk with the
microscope.
 Avoid touching the lenses of the microscope. The oil and dirt on your fingers can
scratch the glass.
 Clean smudges on the microscope glass with a piece of lens paper. Any other
material, such as tissues or even cotton, can scratch the glass of the microscope.

6. Laminar flow
 Laminar flow Also known as streamline flow
 Occurs when the fluid flows in parallel layers, with no disruption between the
layers
 The opposite of turbulent flow (rough)
 In fluid dynamics (scientific study of properties of moving fluids), laminar flow
is:
 A flow regime characterized by high momentum diffusion, low momentum
convection, pressure and velocity independent from time.
• momentum diffusion refers to the spread of momentum (diffusion) between
particles of substances, usually liquids

7.  Laminar flow over a flat and horizontal surface can be pictured as consisting of
parallel and thin layers.
 Layers slide over each other, thus the name ‘streamline’ or smooth.
 The paths are regular and there are no fluctuations.

8. Uses
• Laminar Flow Cabinets are suitable for a variety of applications
• where an individual clean air environment is required for smaller items, e.g.
particle sensitive electronic devices.
• In the laboratory, Laminar Flow Cabinets are commonly used for
specialised work.
• Laminar Flow Cabinets can be tailor made to the specific requirements of
the laboratory
• ideal for general lab work, especially in the medical, pharmaceutical,
electronic and industrial sectors.

9. VACUUM PUMP
 A vacuum pump is a tool that eliminates gas molecules from a sealed volume
so as to leave behind a partial vacuum. It removes gas from an area, leaving a
partial vacuum behind.
 It also will remove water from one area to another, exactly like sump pump
does In a basement.
 They are utilised in an industrial setting in order to produce vacuum tubes and
electric lamps, and to process semiconductors.
 The pumping speed is a very important factor in measuring its performance.
Pumping speed means the volume flow rate of a pump at its inlet.

10. Uses
 To provide suction to drive the aspiration or filtration of liquid or
suspended samples
 To induce or control solvent evaporation by reducing vapor pressure, as in
ovens, rotary evaporators, gel dryers, and concentrators
 To improve instrument-detection sensitivity by evaluating air molecules
that might obscure or contaminate samples, as in a mass spectrometer
 To collect gas samples from test chambers or the atmosphere
 To provide a negative pressure (that is, less than atmospheric pressure)
environment to prevent escape of potentially hazardous sample materials.

11. VISCOMETER
 Viscometers are used to measure viscosity in most circumstances.
 Also known as “VISCOSIMETER.”
 They work for fluids whose viscosity does not change under varying flow
conditions.
 The measure of the resistance is taken by measuring the drag resistance
during relative motion Through the fluid.

12. Applications of viscometers
• Viscometers have wide and varied applications in the materials science and
chemical industries for materials such as oil, asphalt, plastics, wax, paints,
coatings, and adhesives.
• They are also used for food and beverages and personal-care products such as
cosmetics, shampoo, and toothpaste.
• Measuring viscosity is important when considering process conditions for
materials that need to be pumped or piped. Viscosity also affects dipping and
coating performance, which is particularly relevant to paints and inks.
• Viscosity is a very useful indirect measure of material properties including
molecular weight and density, both of which affect flow behavior.
• Viscometers can therefore be used to monitor batch consistency and quality
control.

13. Thermometer
 Laboratory thermometers are devices used to measure temperature. There are
many types of lab thermometers such as differential, mechanical, logging, etc.
 Laboratory thermometers are progressively providing digital reading displays
and are input-capable to computer and software programs for logging
purposes.
 A laboratory thermometer can be used for a number of scientific applications
and can be found across nearly all laboratories especially in pharmaceutical,
environmental, food, and petroleum testing.
 While a lab thermometer can measure how hot or cold a sample or
environment is, the range of measurement can vary widely between models.

15. Media
A growth medium or culture medium is a solid, liquid or semi-solid designed to
support the growth of microorganisms or cells.
Composition :-The general composition of a medium is as follows:
 H-donors and acceptors (approximately 1-15 g/L)
 C-source (approximately 1-20 g/L)
 N-source (approximately 0.2-2 g/L)
 Other inorganic nutrients e.g. S, P (50 mg/L)
 Trace elements (0.1-1 ug/L)
 Growth factors (amino acids, purines, pyrimidine’s, occasionally 50 mg/L,
vitamins occasionally 0.1-1 mg/L)
 Solidifying agent (e.g. agar 10-20 g/L)
 Solvent (usually distilled water)
 Buffer chemicals

16. Preparation of Media :-
 Required amount of chemicals is dissolved in distilled water using
Erlenmeyer flasks or rimless clean test tubes.
 pH is adjusted by adding 1N of NaOH or N/10th of HCI
 15ml capacity of test tube ->5ml of liquid medium is taken
 250ml flask-> 100ml of media
 These are plugged with non adsorbent cotton and are covered using brown
paper.
 This is kept for autoclaving - sterilization done at 15lbs/inch^2 at a
temperature of 121 degree Celsius for 15min
 Later cooled down and poured in petri plates or test tubes which solidifies
to form a medium.

17. Eg.- PREPARATION OF NUTRIENT AGAR MEDIA:-
Nutrient Agar is a general purpose, nutrient medium used for the cultivation of
microbes
Composition of Nutrient Agar
 •0.5% Peptone
 •0.3% beef extract
 • 1.5% agar
 • 0.5% NaCl
 •Distilled water
 • pH is adjusted to neutral (7.4) at 25 0C

18. Preparation of Nutrient Agar
 Suspend 28 g of nutrient agar powder in 1 liter of distilled water.
 Heat this mixture while stirring to fully dissolve all components.
 Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
 Once the nutrient agar has been autoclaved, allow it to cool but not solidify.
 Pour nutrient agar into each plate and leave plates on the sterile surface
until the agar has solidified.
 Replace the lid of each Petri dish and store the plates in a refrigerator.

19. METHOD OF STERILIZATION
STERILIZATION :-
• The process of killing or removing bacteria and all other forms of living
micro-organisms and there spares from preparation.
• Essential concept in the preparation of sterile pharmaceutical products
• Its aim :- is to provide a product that is safe and eliminates the possibility of
introducing.

20. Physical method
1. DRY HEAT STERILIZATION
Instrument- 'OVEN' : - specially designed instrument - electrically heated and
thermostatically controlled. Expose at 160 °C for 1 hour.
Advantage- it is suitable method for sterilization of substances destroyed by
moisture.
Disadvantage- oven long heating time, high temperature.
2. MOIST HEAT STERLIZATION
Instrument- ‘AUTOCLAVE’
Heating process in autoclave - saturated steam under pressure is allowed to
penetrated through materials for 15 minutes and temperature 1210C.

21. Advantage- micro organism are killed most efficiency in lesser time due to
high pressured saturated steam.
Disadvantage- unsuitable for materials not withstanding temperature of
115°C or more during heating.
3. STERELIZATION BY RADIATION
Two techniques involved:
I. Alteration of chemicals lead to form new compound in cells destroying
the micro-organism itself.
II. Vital structure like nuclear protein are destroyed killing the micro-
organism.
e.g., Co-60 - used for gamma ray sterilization process.

22. Gamma rays –
 generally obtained from radio isotope(Co-60) during disintegration of
unstable atoms .
 kill micro-organisms by isolating atoms of essential substance of cells
present in them.
Advantages:-
1. No significance rise in temperature
2. Continuous process due to short exposure time.
Disadvantages:-
1. May lead to color change.
2. Solubility of preparation leading to decomposition of certain materials.

23. MECHANICAL METHOD
The solution to be sterilized is passed through depth- filter or screen- filter
which includes -
1. Particulate filters
2. Microbial filters
3. Final filter
• Pharmaceutical solutions are sterilized by this method.
• The micro-organism are physically removed by absorption on the filter
medium or by mechanism.
• Filtration filling and sealing processes are under a septic condition.
• Sterilization test must be done.

24. CHEMICAL METHOD
Gaseous sterilization-
 Ethylene oxide used.
 Special type of chemical sterilization using gases and vapour
 The gas used is safe & non-inflammable.
 Now-a-days, ethylene oxide most widely used gaseous sterilization agent in medical
science.
Advantages:-
• It has penetration power quite useful for sterilizing surgical instruments (such as
catheter, needles, plastics, disposables)
Disadvantages:-
• Very slow sterilization process
• Very costly equipment

25. DISINFECTION
 Decontamination - removal of microorganisms contaminating an object.
 Preservation - preventing methods of microbes-caused spoilage of
susceptible products(pharmaceuticals, foods).
 Sanitization - removal of microbes that pose a threat to the public health,
food industry, water conditioning.
 Chemical agents :- Alcohols, aldehydes, halogen, phenols, surfactants,
heavy metals.
e.g., ethylene oxide – most commonly used for sterilisation.
 Advantages:-1. Widely used in hospitals for materials that cannot
withstand steam sterilisation.
 Disadvantages:- 1. 40-60% humidity in sterilizing chamberanitizer-an
agent, usually a detergent, that reduces the numbers of bacteria to a safe
level.

26. Thank You