ASEPTIC TECHNIQUES IN THE LABORATORY

aseptic techniques

refer to any method used to sterilize and maintain the sterility of an

object or location, such as the laboratory

sterilization

complete killing of all forms of microorganisms, including bacterial spores

disinfection

killing or removing of harmful vegetative microorganisms.

disinfectant

chemical substance used to achieve disinfection.

antiseptic

disinfectant that can be safely used on living tissues

heat

Most important physical method that should be used whenever possible

160C, one

Dry heat at temperature of — for — hour

121 or 134C, 15 minutes

Moist heat e.g. Autoclave at — for — or more

dry heat

kills microorganisms by destroying their oxidative processes

naked flame

Simplest method of dry heat is exposing item to be sterilized to the —

• e.g Bunsen burner- for sterilizing bacteriological loops, knives, blade

hot air oven

Has electric element in chamber as source of heat plus a fan to circulate

air for even distribution of heat in chamber.

• Used for:

- Metals

- Glassware

direct flaming

whenever rapid and repeated sterilization is required, the simplest method is —

metal instruments, platinum wire loop, forceps and scissors

direct flame is used for — such as —

bunsen burner

is commonly used for sterilization by dry heat (direct flame)

hot water

Moist heat uses —

denaturating proteins

Moist heat kills microorganisms by —.

autoclaving

standard sterilization method in hospitals (standard method)

>100C

Autoclave works under the same principle as the pressure cooker where water boils at increased atmosphere pressure, because of increase pressure the boiling point of water is

pure saturated steam under pressure

The autoclave is a tough double walled chamber in which air is replaced by —

saturated steam

The air in the autoclave chamber is evacuated and filled with —

chamber

The — of autoclave is closed tightly the steam keeps on filling into it and the pressure gradually increases creating saturated steam.

• Pressure: 15 psi (pounds per square inch)

• Temperature: 121 degrees Celsius

• Time: 15 - 30 minutes

Major factors for effective autoclave:

physical method

Monitoring of Autoclaves

• use of thermocouple to measure accurately the temperature.

Monitoring of Autoclaves

chemical method

Monitoring of Autoclaves

• it consists of heat sensitive chemical that changes color ar the right temperature and exposure time

biological method

Monitoring of Autoclaves

• where a spore-bearing organism is added during the sterilization process and then cultured later to ensure that it has been killed

cotton plugs

are used to stop air movement that might carry microbes.

culture medium

is a nutrient solution used to grow microorganisms.

identified

▪ Bacteria have to be grown (cultured) for them to be —.

isolated

▪ By appropriate procedures, culture media have to be grown separately (“—“) on culture media and obtained as pure for study.

louis pasteur

The original media used by —

urine or meat broth

The original media used by louis pasteur

diffuse growth

Liquid medium –

discrete colon

Solid medium –

• Robert Koch

water

role in solubilizing nutrients, transporting them and ensuring hydrolysis reactions

water, carbon source, nitrogen, mineral salts

In order to grow, bacteria need a minimum of nutrients:

carbon sources

It is essential for bacteria to produce carbon molecules, such as fats,

carbohydrates, proteins and nucleic acids. Bacteria can use inorganic carbon sources, such as carbon dioxide, or organic sources such as sugars and alcohols

nitrogen

Bacteria require large amounts of — for synthesis of all the key constituents of the cell, including amino acids, pyrimidines and purines, NAD, and amino sugars

mineral salts, phosphate, sulphate, magnesium or calcium, and vitamins

examples of micronutrients needed for bacteria

colony

macroscopically visible collection of millions of bacteria originating from a single bacterial cell.

cooked cut potato

earliest solid medium by robert koch

frau hesse

he discovered agar

2% agar

it is Used for preparing solid medium

seaweeds

agar is obtained from —

98c, 42c

agar melts at — and sets at —

solid media

Types of culture media based on consistency

• contains 2% agar

• colony morphology, pigmentation, hemolysis can be appreciated

liquid media

Types of culture media based on consistency

• no agar

• For inoculum preparation, Blood culture, for the isolation of pathogens from a mixture

semi solid medium

Types of culture media based on consistency

• 0.2 - 0.5% agar.

• It’s mainly used to study the motility of microorganisms, distinguish between motile and non-motile bacterial strains

simple media / basal media

Types of culture media based on constituents/ ingredients

• Eg: NB, NA

• NB consists of peptone, meat extract, NaCl,

• NB + 2% agar = Nutrient agar

enriched media

Substances like blood, serum, egg are added to the basal medium.

▪ Used to grow fastidious bacteria that are exacting in their nutritional needs.

▪ Eg: Blood agar, Chocolate agar

complex media

Types of culture media based on constituents/ ingredients

• Media other than basal media.

• They have added ingredients.

• Provide special nutrients

  ▪ E.g.: MacConkey agar, Tryptic soy agar, Chocolate agar

synthetic or defined media

Types of culture media based on constituents/ ingredients

• Media prepared from pure chemical substances and its exact composition is known

  ▪ Eg: peptone water – 1% peptone + 0.5% NaCl in water

fastidious

microorganisms with complex nutritional require

enrichment media

Liquid media used to isolate pathogens from a mixed culture.

inhibitory substances

enrichment Media is incorporated with — to suppress the unwanted organism.

selective media

The inhibitory substance is added to a solid media.

mac conkey’s medium

selective media for gram negative bacteria

thiosulfate-citrate-bile salts-sucrose

selective media for V. cholerae

löwenstein-jensen medium

selective media for M. tuberculosis

wilson and blair medium

selective media for S. typhi

potassium tellurite medium

selective media for diphthreia bacilli

sodium salts

to select halophilic bacteria that resist very high amounts of salts

chemical substances

These inhibiting substances include potassium tellurite and bile salts, which inhibit Gram-positive bacteria, or lithium chloride which eliminates Gram-negative bacteria

dyes

used as a colour indicator in a culture agent against certain bacteria.

crystal violet

is one of the most commonly used dyes to inhibit bacteria.

malachite green and methylene blue

are also used to inhibit Gram-positive and Gram-negative bacteria and Gram-positive bacteria, respectively

indicator media

These media contain an indicator which changes its colour when a bacterium grows in the

pink colonies

Mac Conkey Agar

▪ Lactose fermenters —

colourless colonies

Mac Conkey Agar

▪ Non-Lactose fermenters

transport media

Media used for transporting the samples.

▪ Delicate organisms may not survive the time taken for transporting the specimen without a transport media.

stuart’s medium

non nutrient soft agar gel containing a reducing agent

anaerobic media

these media are used to grow anaerobic organisms

• eg: Robertson’s cooked meat medium, thioglycolate medium