Microbiology Laboratory: Introduction to

Media & Equipment

Diana Verano, M.S.

Pamantasang ng Lungsod ng Maynila
College of Nursing
DEFINITION OF TERMS*
Culture Medium: (singular):
nutrient material prepared for the growth of microorganisms in a

laboratory

Media: plural

Inoculum: microorganism introduced into a culture medium to

initiate growth

Culture: microorganism that grow and multiply in and on a culture

medium

* Derived from Tortora et al., Medical Microbiology

DEFINITION OF TERMS*
Sterile
 material devoid of any living microorganism and viruses

Agar: A complex polysaccharide and used as a solidifying agent

added to the solid medium from marine alga. Solidifies at 40 ºC

* Derived from Tortora et al., Medical Microbiology

 CULTURE MEDIA IN POWDER FORM
(NON-STERILE)

Left photo: Media bottle

Bottle Plastic type Right photo: Powder form of Media
Both for Solid medium & Broth
Media Name: Nutrient Agar
DEFINITION OF TERMS
Sterilization
 process of destruction or removal of all forms of microorganisms

Substrate
 material that a microorganism feeds on
(natural or artificial ex: culture medium)

Enzymes: made up of proteins that catalyzes chemical reactions.

Culture Media can be solid or liquid

Left broth in tubes Right: TSI agar in tubes (butt slant)

Culture Media: solid or liquid in Erlenmeyer Flask

Flasks with Cotton Plug

Left: broth media

Right: Solid media, should be poured Autoclavable plastic cap

at 42 – 45 ºC into Petri dish
Photo from: semanticscholar. org
CULTURE MEDIA
Solid medium: in test tube
and petri dish

 slants:solid agar in test

tubes

 Deep: agar solidies in a

vertical tube

 Culture medium to solidify:

Agar should be at 1.5-2%
CULTURE MEDIA

 Solid media: useful for identifying organisms based on

colony characteristics

 Colony: The bacterial population is considered derived

from 1 bacterial cell

Liquid broth: only in test tubes

BROTH

 media nutrients are added in

water

 Microbial growth: cloudiness

or turbidity

 Turbidity is measured by
spectrophotometer called as
optical density

o Seen by naked eye: Turbidity,

1x 106 bacteria cells Left turbid Right:clear
CULTURE MEDIA
Media components: (depends upon the media)
Nutrients:
 Examples:carbohydrates,serum,whole blood,yeast
extract,peptone,beef extract

 Solidifying agent: Agar

 Water
 Example: distilled water

 Additives or supplemets
 Examples: dyes,vitamins,amino acids,growth factors and
antibiotics
PROPERTIES OF AGAR
 no substitute

 Few microorganisms can degrade,remains solid

 Liquefies at 100ºC

 Solidifies at 40ºC and below

 from red algae Gelidium (Rhodophyta)

 Mostly made up of galactose

Lab experiments
 after reaching 100ºC,remains liquid even between 42-45ºC
SPORE FORMERS
Gram-positive bacteria members of Genera
Bacillus and Clostridium form vegetative
cells and endospores

Endospores survives at:

 resistant to chemicals,

 boling point of water at 100 ºC, low

temperature, irradiation
 extreme pH conditions

 Even with low water availabiliy

 Contains calcium dipocolinate in the core

 Only destroyed by direct heating and

autoclaving process
SPORE FORMERS
 Genus Bacillus members are
aerobic, Genus Clostridium
members are anerobic

 Both Genera members are

found in soil and endospores
can be spread by the wind

 Bacillus members are

common contaminants in the
lab
Laboratory Apparatus, Glasswares &
Equipment
 TEST TUBES
with cotton plug

with sterilizable screw cap

Petri dish: without culture medium
Petri plate: with culture media
Parafilm TM (brand name) to seal individual Petri plates,
ready for storage.
Sterile disposable, plastic type petri dish.
Used only 1x.
Once the plastic cover is opened, all petri dish should be used
Non-reusable, not autoclavable
 Analytical Balance & Triple Beam Balance to measure Media powder
* Always calibrate first before using
 COVERING WITH ALUMINIMUM FOIL
(BEFORE AUTOCLAVING & AFTER STERILIZATION/STORAGE)

*Metal test tube rack: autoclavable

Autoclavable plastic test tube rack, also in different colors
INOCULATING LOOP AND NEEDLE
 Used for transfer of inoculum to
test tubes and petri dishes

 Made up of platinum or nichrome

wire

 Should be initially sterilized upon

use

 Inoculating loop:used in broths

and solid media

 Colored types: plastic,

disposable, used only 1x
INOCULATING LOOP AND NEEDLE

 Inoculating needle: for

solid media only using
stab method

 Should be sterilized by
heating almost vertically in
a flame

 Color: red hot (sterilized)

ALCOHOL LAMP

 Sterilizes inoculating loops and

needles

 Used in Aseptic Technique of

Inoculum transfer on test tubes and
dispensing of media on petri dish
from Erlenmeyer flask.
BACTI-CINERATOR
STERILIZER

 Sterilizes inoculating
loops and needle up to
10 seconds

 Kills microorganisms at
815 ºC

 Using Infra-red Heat

AUTOCLAVING: ROUTINE STERILIZATION
PROCESS FOR MICROBIOLOGY LABORATORY

Autoclave
 specialized pressure cooker

 Uses moist heat

For:
 non-heat labile substances Ex: media
 heat stable solutions using steam

 Sterilization of glasswares & small metal

instruments

 Sterilizes materials at pressure of 15psi for

15 minutes or 121ºC for 15 minutes

 Endospores & viruses are destroyed

MOIST HEAT: physical control of microbial
growth

moist heat: water vapor aids because

of high penetrating property

Proteins in cell undergo denaturation

at high temperatures: process of
clumping and changes in shape

Automated Autoclave
AUTOCLAVING: ROUTINE STERILIZATION
PROCESS FOR MICROBIOLOGY LABORATORY
Substances not to be autoclaved

 Heat susceptible products (majority

of plastics if thin)

 Paper

 chemicals like enzymes

 Inoculum needed for experiment

Left photo: autoclave/sterilizer in clinics
Right photo: Retort, bigger scale autoclave used in manufacturing
DECONTAMINATION USING MOIST HEAT

 Separate Autoclave Equipment is used

 Using 15psi for 15 minutes or 121ºC

for 15 minutes

 Endospores & viruses are destroyed

 petri plates, test tubes with culture

before discarding

 Other lab materials for reuse: pipette

tips
WATER BATH

Water bath: maintains temperature settings,

Pour non-sterilized water inside, keeps media liquefied

Why does it need a lid if you placed a sterile media?

HOT PLATE: MELTING & MIXING FLASKS OF MEDIA & LIQUIDS

Left photo: Hot plate

If your sterile solid media solidified: use Hot plate to melt agar
Improvised Water bath: to melt sterile media easier ( gas stove + metal container + water heated
to boiling

For mixing media still prepared for sterilization (non-sterilized): insert magnetic stirring bars (Right
Photo) then heat with hot plate
Vortex mixer: mixers of test tubes or conical centrifuge tubes
To ensure solid particles into homogenous mixture
Centrifuge: equipment that separates solids particles from liquid
For medical tests: blood & its component, urine and it contents for
analysis in the pathology lab in hospitals.
Blood Collection Tubes, color coded for different blood tests
OVEN
 Uses Dry heat

 Uses hot air, removes

moisture
 Proteins in cell undergo
denaturation at high
temperatures: process of
clumping and changes in
shape, leads to cell death
 Sterilizes materials at

160 –180 °C for 2-3 hours

 for glass materials
(example:
petridishes,pippettes)
OVEN
 Made up of an insulated
cabinet

 Thermometer monitors
the temperature
 The shelves are perforated
so there is a proper
circulation of air
INCUBATORS
 Maintains constant
temperature

 Similar to hot oven

but the temperature is
maintained by the
automatic device
called thermostat

 Inocula are cultured

at optimum
temperature Ex: 37°C
 BIOLOGICAL REFRIGERATOR
 Stores microbial culture up to 2 yrs

 Sterile media for storage

 Culture collection are usually stored

in a refrigerator to avoid dehydration
of cells/ drying up

 Largest Culture Collection: UPLB

BIOTECH

 Other laboratories in schools,

government research labs,
manufacturing companies also keep
culture for storage

 Don’t place food beside culture.

 SHAKER

Rotary Shaker moving flasks in circulatory movement in rpm (revolutions

per minute)

What is lacking on the Erlenmeyer flask if it is shaking?

MICROPIPETTES

 Precision instruments

 They are provided with plastic

tips for dispensing liquid

 micropipette tips are usually

packed in autoclavable plastic
boxes
MICROPIPETTES
 They are provided with plastic
tips for dispensing liquid

 Blue tips:100µL to 1000µL

 1000µL = 1mL

 yellow tips:10µL to 100µL

 white tips:1µL to 10µL

ELECTRONIC PIPETTE
CONTROLLER
 Needs glass pippette

 Used to transfter media to test tubes

(non-sterile)

 Pipettes media thru pushing button

 handling sterile media for dispensing in

petri dish: glass pipette should be
sterilized, this equipment, sanitized with
alcohol, then aseptically transfer media

 Petri plate: contains 20- 25 mL medium

SPECTROPHOTOMETER

 Microbiologists use this

instrument to measure turbidity,
represented by OD (optical
density)

 Higher OD, more cells, more

turbid
 Analyzed samples are placed in
small cube tubes called cuvette.

 small test tubes can be used

only if the size of the test tube
fits
GLASS SLIDES & COVER SLIP

 Sterilize glass slide under flame of alcohol

lamp or place in a Petri dish in an oven for
2hrs.

 Avoid bubble formation while covering with

cover slip.

 Forceps can be used in handling sterile

glass slides
Dropper & Glass Pasteur Pipette for sterile water transfer to glass slides
Filter Sterilization by Syringe Filter
Filter size: 0.2 µm or 0.45 µm
Remove the needle from syringe, place the syringe filter
The filtered liquid is particle & bacteria free
Place the liquid in a sterile container (tube)
 VACUUM FILTRATION SET-UP: FOR VIRUSES
(PHAGES)

Broth media containing phages + solvent :

Filter paper is placed on Buchner funnel
Filter paper separates solid particles with liquid
faster rate with Vacuum pump creates vacuum to Pull solvent & liquid away from funnel
Filtered liquid contains viruses
Anaerobic jar and Candle jar: to cultivate anaerobic microorganisms
Presence of Oxygen kills obligate anaerobes
BIOLOGICAL SAFETY CABINET (BSC)

Prevents contamination and

protects the operator from
exposure from aerosol
exposure to infectious agents

Air is sterilized by
UV light
Filtration by HEPA filter
HEPA filter: 0.3 µm

Particles
larger than 0.3 µm
are removed
BSC class 2
BIOLOGICAL SAFETY CABINET (BSC)
 Categorized by class according
to the dgree of containment they
afford

Class 1
 Sterilizing only the air to be
exhausted

 Class 2
 Also called laminar flow BSC
 Sterilize the air over the
infectious material and air to be
exhausted
 Most hospital labs use this type
BSC class 2
BIOLOGICAL SAFETY CABINET (BSC)
Class 3

 Completely enclosed
 With negative pressure
 With rubber gloves
 Offer Most protection

BSC class 3
 MEASUREMENT OF MICROBIAL
GROWTH
Microbial growth can be measured through:

1.Total cell count

 microscopic cell count (hemocytometer/haemocytometer)

 Dead+ living cells counted

2.Viable cell count or plate count

 Each viable cell can grow and divide to form a colony are
counted, expressed as colony-forming units (cfu)

 Only living cells capable of forming colonies are counted

200 -250: counted cells (TNTC)
Too numerous to count

Food /drug sample: unacceptable with

TNTC

 PCA (plate count agar) and Nutrient Agar usually used as

media
HEMOCYTOMETER
 Covered with a cover slip, using
micropipettor, dislodge a small drop of
sample in a well in hemocytometer

 Look under the microscope

 2 counting chambers, located at the center.

 Count all cells in 4 squares, then get

average by dividing into 4

Computation of No. of cells/mL

Example: 20 average cells x 104 = 200,000 or
2 x 10 5 cells / mL
Colony counter equipment: Manually count colonies with a grid
Or Computer Assisted Colony Counter
MEASUREMENT OF MICROBIAL
GROWTH
Microbial growth can be measured
through:

3.Turbidity Method
 Estimating microbial growth
 Use spectophotometer as
optical density (OD)

Spectrophotometer:
measures unscattered light

 High OD,more turbid,more cell

number

 Turbidity: cloudiness of broth

 OD should be obtained with Left tube: turbid Right tube:clear

viable count and total cell count