Microbiological instruments

Autoclave Check autoclave for any items left from the Autoclave operates on the principle of moist
previous cycle. heat sterilization.

Add sufficient water to the chamber Steam under pressure is used to sterilize the
materials inside the chamber.
Place materials to be sterilized inside the chamber
High pressure increases the boiling point of
Close the lid and tighten screws for an airtight water, achieving a higher sterilization
condition temperature.

Switch on electric heater Water's boiling point increases under higher

pressure.
Adjust safety valves to maintain required pressure
High pressure aids rapid heat penetration into
Allow air-water mixture to escape through
material.
discharge tube until no more bubbles come out
Moisture in steam coagulates proteins, causing
Close drainage pipe
irreversible loss of microbial function.
Allow steam to reach desired pressure (usually 15
Autoclave operates at 121°C and 15 psi pressure
psi)
or 775 mm Hg.
Whistle blows to remove excess pressure
Steam kills microbes upon contact, giving off
Run autoclave for holding period (usually 15 latent heat.
minutes)
Condensed liquid ensures moist killing of
Switch off electric heater microbes.

Allow autoclave to cool until pressure gauge Sterilization duration varies based on material
indicates atmospheric pressure contamination level.

Open discharge pipe to allow entry of air Pressure release occurs through a whistle once
sterilization is complete.
Open lid and remove sterilized materials
Chamber pressure returns to ambient while
contents remain hot.

Hot air oven Plug in and switch on the oven. Hot air oven principle: dry air sterilization
through convection, conduction, and radiation.
Preheat the oven for 30 minutes.
Heating elements warm air inside chamber.
Set the temperature gauge according to the
 volume of items to be sterilized. Fans circulate air evenly, exposing sample
surfaces to hot, dry air.
Load items onto shelves with proper spacing for
heat circulation. External surface of items heats up, heat
transfers towards item's center via conduction.
Close the door securely.
Heat causes water inside microorganisms to
Monitor temperature with a thermometer. evaporate.

Switch off the oven when holding time is reached. Results in oxidative damage to cellular
constituents, protein denaturation, elevated
Allow the oven to cool before opening the door.
electrolyte levels, and microorganism death.
Remove samples using oven mitts or tongs.

Close the door after removing samples.

LAF Check the laminar flow cabinet for any items Principle of laminar flow cabinet: based on
susceptible to UV rays. laminar flow of air through the cabinet

Close the glass shield and switch on the UV light Works by inwards flow of air through HEPA
for 15 minutes for surface sterilization. filters to create particulate-free environment

Turn off the UV light and wait for 10 minutes Air passes through filtration system, exhausted
before switching on the airflow. across work surface as part of laminar flow

Turn on the airflow about 5 minutes before Air first passes through filter pad or pre-filter
beginning operations. for streamlined flow

Open the glass shield and switch on the Blower or fan directs air towards HEPA filters
fluorescent light during operation.
HEPA filters trap bacteria, fungi, and other
Optionally, sterilize the working bench with particulate materials
disinfectants like 70% alcohol for added
protection. Effluent air passes through perforation at
bottom rear end of cabinet
After completing work, turn off the airflow and
fluorescent lamp, and close the glass shield. Most air passes over working bench towards
operator's face

Laminar flow hood enclosed on sides, maintains

constant positive air pressure to prevent
contaminated air intrusion

Centrifuge - Centrifuge utilizes sedimentation principle via

gravitational force.

- Centrifugation technique employs centrifugal

field to separate particles in liquid medium.

- Particles placed in centrifuge rotor, typically in

 bottles or tubes.

- Sedimentation: gravity causes particles to

separate from fluids.

- Suspended substance may include powder or

clay-like particles.

- Simple filtration filters particles larger than 5

micrometers.

- Particles less than 5 micrometers exhibit

Brownian motion and don't sediment under
gravity.

- Central force aids in separating these particles.

Incubator - Check for any remaining items from previous - Incubator principle: Microorganisms require
cycles before using the incubator specific conditions for growth.

- If multiple organisms require the same - Parameters: Temperature, humidity, oxygen,

parameters, they can be placed together in the carbon dioxide levels.
same incubator
- Thermostat and thermometer: Maintain
- Close the door of the incubator and switch it on constant temperature, readable externally.

- Heat the incubator to the desired temperature - Heating and no-heating cycles: Control
using a thermometer to monitor temperature within the incubator.

- Set specific parameters such as CO2 - Insulation: Creates isolated conditions for
concentration and humidity if required effective growth.

- Place petri dish cultures on perforated shelves - Maintenance of humidity and airflow: Mimic
upside down to prevent condensation natural environment for organisms.

- Seal petri dish cultures with adhesive tapes or - CO2 concentration adjustment: Balances pH
place them in plastic bags/containers for longer and humidity for growth.
incubation periods
- Variation: Shaking incubator for continuous
- Lock the door and leave the plates inside for the movement of cultures, aiding aeration and
required time before removal solubility studies.

Rotary Shaker

Colorimeter - Rotate Power Switch knob clockwise to switch on - Incident light beam (Io) passes through a
device solution, reflected (Ir), absorbed (Ia), and
transmitted (It).
- Allow 15 minutes for colorimeter warm-up
- Colorimeter measures Io, eliminating Ir to
- Turn Wavelength Control knob to desired
wavelength
 - Press MODE until "Transmittance" light turns on calculate Ia.

- Use Zero Control knob to set T-factor to 0.0% - Based on Beer-Lambert’s law: A ∝ cl, where A
= Absorbance, c = Concentration, l = Length, ∈ =
- Ensure sample chamber is empty and cover is Coefficient of absorption.
closed
- Colorimeter uses lenses to guide light through
- Place blank solution in cuvette up to triangle the solution, comparing color to a standard.
mark
- Microprocessor calculates absorbance/%
- Wipe cuvette exterior with Kimwipe to remove transmittance by comparing light before and
fluids/fingerprints after the solution.

- Insert cuvette into chamber with guideline facing - Calibration curve plotted using known sample
right concentrations vs. absorbance.
- Align cuvette guideline with sample chamber - Unknown sample concentration determined
guideline by rotating clockwise by comparing results to calibration curve.

- Set display to 100.0% using

Transmittance/Absorbance knob

- Switch Status Indicator light to read Absorbance

- Adjust display to 0.0 if needed using

Transmittance/Absorbance knob

- Switch display back to Transmittance using MODE

key

- Remove cuvette by rotating 90 degrees counter-

clockwise

- Insert solution cuvette into chamber using

previous steps

- Read %T value directly from digital display

- Select Absorbance using MODE key, then read A

value from display

- Select Transmittance again using MODE key

- Reverse process to remove cuvette from chamber

- Close compartment's cover

pH meter - Ensure all samples reach the same temperature - pH meter works via ion exchange between
sample solution and inner solution of glass
- Use a thermometer to determine sample electrode
 temperatures - pH probe contains sensor electrode and
reference electrode
- Manually enter temperatures into pH meter or
use ATC probe - Sensor electrode bulb has porous glass
membrane coated with metal salts and silica
- Uncover sample beakers and prepare samples
- Hydrogen ions from sample solution replace
- Rinse pH electrode with deionized water before metal ions around sensor electrode bulb
use
- Reference electrode maintains constant
- Rinse electrode over waste beaker to prevent voltage regardless of pH changes
sample contamination
- pH meter converts electric flow into pH value
- Do not use sample beaker for rinsing electrode by comparing generated voltage to reference
electrode
- Insert electrode into first sample with tip and
junction submerged - Increase in acidity raises voltage and lowers
pH reading
- Stir sample moderately and uniformly
- Increase in alkalinity or concentration of
- Set meter to begin reading
hydroxyl ions reduces voltage and raises pH
- Wait 1 to 2 minutes for stable pH and reading
temperature reading

- If more samples, repeat steps 3-6

- Submerge electrode in each sample to same

depth for accuracy

- Clean electrode with deionized water after

measurements

- Store electrode in pH electrode storage solution

UV chamber

Colony counter - Petri plate placed on electronic pressure pad

with light illumination

- Each colony marked by tapping plate with

auto marker probe pen

- Count registered in digital display by touch

pressure

- Pressure can be adjusted to avoid overlooking

or counting colonies twice

- Equipment includes graticule for Wolfheugal,

segmentation disc, and centering adapters for
50-90mm plates

- Additional features: darkening background for

transparent colonies, glare-free illumination for
optimal peripheral colony viewing

- Integrated averaging tool for multiple plate

counting