Pure Culture Techniques

Bolo, Diamos, Gonzales, Pilapil, Oliva

By PresenterMedia.com

Introduction
Microbiology

The branch of biology that deals with microorganisms.

Introduction
In order to be able to study the microorganisms, we need to isolate them in the laboratory and prepare them in pure cultures. In the natural environment, microorganisms form a mixed culture.

Introduction
Microorganisms could be: Pathogenic Staphylococcus aureus Non-pathogenic Lactobacillus casei Opportunistic Escherichia coli

Introduction
Pathogenic

Microorganisms that cause diseases and health problems e.g. Staphylococcus aureus

Introduction
Non-pathogenic

Microorganisms that does not impose any health problems e.g. Lactobacillus casei

Introduction
Opportunistic Microorganisms that only cause diseases upon weakening of the immune system e.g. Enterobacter aerogenes

Introduction
Microorganisms normally found to live on or within the body of an animal or a plant is called a Normal Flora e.g. Propionibacterium acnes

Introduction
Different methods of preparing a pure culture: Capture Plate Method Swab Inoculation Method Streak Plating Surface Plating Pour Plating or Miles and Misra Method

Introduction
Streak Plating The method into which a colony of a single species of desired microorganism is obtained by primarily spreading or scattering of cells.

Introduction
Streak Plating

Introduction
Surface Plating Employs the use of hardened and pre-poured agar with dry surfaced plates. Diluted specimens are planted into the surface of agar with the use of bent glass rods. Method of choice when identifying microorganisms and using selective media.
NOTE: Strict aerobes are cultured strictly by surface plating method

Introduction
Pour Plating or Miles and Misra Method A technique in obtaining a pure culture wherein the number of colony forming units of a bacterial suspension in a media is determined.

Introduction
Slide culture technique is a method into which well-representing characteristics of the microorganism can be viewed under the microscope that would later lead into its identification and characterization.

Objectives
RECOGNIZE

Students should be able to:

different types of microorganisms present in our everyday environment different types of methods to isolate microorganisms from the environment

CARRY OUT
OBTAIN

pure cultures of bacteria, yeast and molds

cultural characteristics of bacteria, yeasts and molds common types of preservation methods for bacteria ethical principles observed in handling microorganisms in the laboratory

DESCRIBE

PERFORM BE AWARE

Materials

For this experiment, you would need:

Microscope

Diluent

Distilled Water

Materials

Flasks

Cotton plugs

Materials

Incubator

Lighter

Sterile Glass slide and cotton buds cover slip

Materials

Cedar oil

Glass pipette

Stirring rod

Marker

Materials

Weighing balance

Vortex mixer

Autoclave

Materials

Sterile water

Corn meal agar

Lens paper

Materials

Automated pipettors

Beakers

Stove

Materials

Wireloop and needle

Oven

Colony counter

Materials

L rods

lactophenol Clear nail Sodium hypochlorite polish (or 70% ethanol)

Materials

General purpose medium (on plates)

(in slants)

Bacteria, yeast and mold culture

Isolation of Microorganisms from the Environment Using the Capture Plate and the Swab Inoculation Method
Microorganisms from Air (Capture Plate Method) Microorganisms from Human Skin (Swab Inoculation Method)

Methodology: Part I

Methodology: Part I
Capture Plate/ Gravity Plate Method

Methodology: Part I
Swab Inoculation Method

24-48 hours for NA 4-5 days for PDA

Methodology: Part II
Obtaining Pure Cultures of Bacteria, Yeast and Mold

Streak for Isolation Technique

Methodology: Part II

Obtaining Pure Cultures of Bacteria, Yeast and Mold

Methodology: Part III

Preservation of Microorganisms
Preservation: Mineral Oil

Inoculate pure culture onto fresh media slant.

INOCULATION

INCUBATION
Incubate: 24-48 h ; RT

LAYERING Layer 0.5 inch sterile mineral oil.

STORING Refrigerate (at 4C)

Methodology: Part III

Preservation of Microorganisms
Preservation: Subsequent Transfer

Inoculate pure culture onto fresh media slant.

INOCULATION

INCUBATION
Incubate: 24-48 h ; RT

STORING Store at 4C.

TRANSFER Transfer into fresh slants after one week.

Methodology: Part IV

Preservation of Microorganisms

Cultural Characterization of Bacteria and Yeast

Colony Color Colony Shape Colony Texture

Methodology: Part IV

Preservation of Microorganisms

Colony Morphology
Surface Color Edge Margin Texture

Methodology: Part IV

Cultural Characteristics of Bacteria and Yeast

Methodology: Part IV

Cultural Characteristics of Bacteria and Yeast

Bacillus subtilis in NA.

Bacillus subtilis in nutrient-poor agar.

Methodology: Part IV

Cultural Characteristics of Bacteria and Yeast

Clostridium sp.

Microoccus sp.

Escherichia coli

Mycoplasma pneumoniae

Methodology: Part V

Cultural and Characterization of Molds

Petri dish containing bent glass rod, filter paper, glass slide, two cover slips Place a PDA cube

Methodology: Part V

Cultural and Characterization of Molds Get a loopful of mold colony and deposit along the four corners of agar block Place coverslip on top of block Incubate for a week then observe Note: make sure that the paper will not dry out during incubation period

Slide culture technique

Methodology: Part V

Cultural and Characterization of Molds

Colony color Texture Area of growth Pigmentation Spore arrangement

Methodology: Part V

Cultural and Characterization of Molds

Common molds

Aspergillus sp.

Mucor sp.

Penicillium sp.

Rhizopus sp.