UNIT III: BASIC MICROBIOLOGICAL EQUIPMENT AND TECHNIQUES IN STUDYING

MICROORGANISMS

Topic Outline:

1. Basic laboratory equipment in studying microbes

2. Standard laboratory Practices, General laboratory rules on Aseptic Techniques, Culture Media
Preparation, and Proper Care of Microbiological Equipment
3. Pure Culture: Isolation and Characterization
 Pure Culture
 Instrument used to maintain pure culture
 Aseptic technique
 Pure culture techniques
 Methods of isolation
 Study of pure culture
 Wet mount and Hanging drop method
4. Staining and Microscopy
5. Quantitative Bacteriology
6. Culture Preservation and Maintenance

PURE CULTURE = AXENIC CULTURE

 a culture that contains only one group of microorganisms which is usually obtained when
microorganisms in a culture medium are all of the same species

Brettanomyces bruxellensis on Sabouraud agar

Micrococcus luteus
plate after 11 days

INSTRUMENTS USED TO MAINTAIN PURE CULTURE

1. Pipette – an instrument often used to transfer aliquots of culture, to prepare serial dilutions of
microorganisms, and to dispense chemical agents.

Two types:

a. Blow-out pipette – the final few drops of liquid must be emptied to deliver the correct volume.
b. To –deliver pipette – after the proper amount of liquid has been delivered, liquid will remain in
the tip of the pipette and should not be eliminated.
 2. Inoculating needles and loops
 used to aseptically transfer microorganisms from broth, slant, or agar cultures to other
media.
 both may consist of handles, a shaft, and turret,which holds a nickel-chromium or platinum
wire.

Aseptic Technique

 a set of specific practices and procedures performed under carefully controlled conditions with
the goal of minimizing contamination

Sepsis – a toxic condition resulting from the presence of microorganisms

PURE CULTURE TECHNIQUES

Requirements:

 All apparatus and media must be previously sterilized or freed from microorganisms.
 The desired bacterium must be separated from the naturally-occurring microbial populations
into the sterile medium, and
 Aseptic techniques which prevent the entry of contaminating microorganisms must be
followed.
A. ENRICHMENT CULTURE
 isolation of specific types of microorganisms by a combination of nutrient and physical
conditions.
 used for the isolation of unusual physiological types of microorganisms which are present in
small numbers and which grow slowly
B. SINGLE-CELL ISOLATION TECHNIQUE
 Uses a micropipette or a microprobe to physically pick a single cell and transfer it on an agar
medium.
C. MEMBRANE FILTER TECHNIQUE
 for samples with low population.
o uses a sterile membrane filter having a pore size that retains microorganisms.
D. SERIAL DILUTION
 for samples with low population
o Uses a sterile membrane filter having a pore size that retains microorganism
-3
o Use a series of diluents to thin out microbial population (e.g. dH2O, saline 0.9%, PO4 ,
buffer, etc.)
E. PLATING

Colony = a macroscopically visible (surface or subsurface) growth or cluster of microorganisms on a

solid medium
Plating Techniques:

• Streak Plate

 Key Principle: by streaking, a dilution gradient ins established on the surface of the plate as
cells are deposited on the agar surface.
 Materials needed: wire loop, alcohol lamp

• Spread Plate

 Small volume (0.1ml) of dilute bacterial mixture containing 100-200 cells or less is transferred
to the center of an agar plate.
 Materials needed: L-shaped glass rod, alcohol, alcohol lamp

• Pour Plate

 The original sample is diluted several times to reduce the microbial population sufficiently to
obtain separate colonies upon plating.
 Materials needed: pipettes or micropipette, diluents, alcohol lamp

Study of Plate Culture

Cultural Characteristics:

 Size (measure diameter in mm) –ruler  Margin or edge

 Form  Consistency – doughy, hard, soft, sticky
 Surface – dull, shiny, smooth, glistening,  Pigmentation – color, solubility
powdery  Elevation

Wet Mount

 Consists of a drop or two of the culture placed on a slide and overlaid with a cover glass/slip
 Advantages:
o quick and easy to prepare
 Disadvantages:
o cover glass can damage larger cells
o slide is very susceptible to drying
o can contaminate the handler’s finger

Wet Mount Technique (WMT)

a. Place small drop of suspension on slide.

b. Gently lower coverslip. (Edges touching will spread suspension evenly)
c. Slide ready for viewing

Hanging-Drop Technique (HDT)

 is a special type of wet mount (in which a drop of medium containing the organisms is placed
on a microscope slide), often is used in dark illumination to observe the motility of bacteria
 STAINING AND USE OF THE SLIDE CULTURE TECHNIQUE FOR THE MICROSCOPIC
EXAMINATION OF SELECTED MICROORGANISMS

STAINING TECHNIQUE

 Different Staining Protocols: Bacterial morphology

 Slide Culture Method: Fungal morphology (Molds)

INTRODUCTION

Stained Preparations
Advantages: Disadvantages:
provides contrast more complicated and tedious to prepare
slides can be preserved more expensive
specimens are killed

Smear Preparation

 Smear - a thin dry film of microorganisms

Fixation

 Heat Fixation  Purpose of Fixation

o Direct flame o Kills the cells
o Steam fixation o Makes the cells sticky
 Chemical fixation o Increases apparent diameter of cells
o alcohols

DIFFERENT STAINING PROTOCOLS

1. SIMPLE STAINING (positive and negative)

– only one dye

 Positive/direct cells same color as dye

o Methylene blue
o Crystal violet
o Safranin
  Negative/direct cells colorless or luminous
o India ink
o Nigrosin

 Pairs: diplococci, diplobacilli

 Clusters: staphylococci
 Chains: streptococci, streptobacilli

2. DIFFERENTIAL STAINING (gram staining and acid fast staining)

 Two or more dyes and/or reagents
 Gram Staining: Gram positive or Gram negative
 Acid fast staining: diagnosis of tuberculosis

Gram Staining: Cell Wall

 Cell Walls of Bacteria

o Gram staining helps distinguish between the 2 different types of bacteria
o Gram positive:
 Thicker peptidoglycan walls
 Keeps dark stain
 Appears purple
o Gram negative:
 Thinner cell wall
 Appears pink
 Gram-positive Cell Walls
o Composed primarily of peptidoglycan
o May also contain teichoic acids (negatively charged)
- Help maintain cell envelope
- Protect from environmental substances
- May bind to host cells
o Some gram-positive bacteria have layer of proteins on surface of peptidoglycan

Cell Wall Components:

1. Peptidoglycan 4. Lipopolysaccharide
2. Teichoic acids 5. Braun’s lipoproteins
3. Outer membrane
Correlation of the Gram Stain with properties of bacterial cell walls
Property Gram-postive Gram-negative
Thickness of wall Thick (20-80 nm) Thin(10 nm)
Number of layers 1 2-3
Peptidoglycan (murein) content >50% 10-20%
Teichoic acids in wall Present Absent
Protein/lipoprotein content 0-3% >50%
Lipopolysaccharide content 0 13
Sensitivity to penicillin Sensitive Resistant
Sensitivity to lysozyme Sensitive Resistant
Acid Fast Staining

1. Application of Carbolfuchsin (primary stain)

2. Application of heat (mordant)
3. Application of Acid Alcohol (decolorizer)
4. Application of Methylene Blue (counterstain)

Mycobacteria - group of bacteria with unique lipid fraction called mycolic acid

3. STRUCTURAL STAINING (endospore, capsule, flagella, storage granules)

 Targets specific parts of the cell, allowing the visualization of particular components
o Endospore staining: steam method, cold method
o Capsule staining: negative staining/Duguid’s method, Maneval’s staining, Anthony’s method
o Flagellar staining
o Staining for inclusion bodies: Lipid inclusions, starch and glycogen, metachromatic granules

ENDOSPORE STAINING

 Endospore
o specialized dormant structure (“resting” cells; cryptobiotic) formed by some genera of Gram
positive bacteria (i.e. Clostridium and Bacillus)
o resistant to environmental stresses (i.e. heat, UV radiation, chemical, disinfectants, and
dessication)
o representative almost the entire dry mass of the cell although it occupies only 10% of the
volume of the vegetative cell.
 Spore Structure
o Exosporium – composed of protein
o Spore coat – made up of polypeptide
o Cortex - has dipicolinic acid (DPA, approx. 10% of the dry weight) and Ca (a chelater), has
only 10-30% of the water content of the vegetative cell.
o Core – spore protoplasts surrounded by a usual CW

1. Application of Malachite Green (primary stain)

2. Application of Heat (mordant)
3. Application of water (decolorizer)
4. Application of Safranin (counterstain)

CAPSULE STAINING

 Glycocalyx
o General term for polysaccharide –containing material lying outside the cell;
 Capsule: rigid layers are organized in a tight matrix: macrocapsule and microcapsule
 Slime layer: loosely adhering to the cell surface w/o a clearly defined external border
 Determination of presence of capsule

Techniques:
1. Negative staining
 o simple negative staining
o Quellung reaction: bacterial cells are resuspended in antiserum that contains antibodies
raised against the capsule
2. Chemical fixation and electron microscopy
3. Other staining procedures (considerations: capsular materials are water soluble smears
should not be heated)

 Anthony’s Method
Primary stain: crystal violet
Decolorizing agent & Counterstain: 20% Copper sulfate
 Maneval’s staining
Reagents used:
Congo red (negative charge) – pH indicator 3.0-5.2) –bluish violet to reddish orange
Maneval’s –phenol +CH3COOH +FeCl3 - phenol + FeCl3 (acid fuchsin)

FLAGELLAR STAINING

 Flagella
o Most common organelle for the locomotion of prokaryotic cells
o Stiff (semi-rigid) helical filament (either left-handed or right handed) that rotates like a propeller
o Does not employ fixation by heat and uses clean slides
o Use of freshly prepared chemical mordants like tannic acid or potassium alum to coat the
flagella
o Staining with basic fuchsin or paranosaline for visualization

Staining Storage Granules

 Storage Granules
o Distinct granules that may occupy a substantial part of the cytoplasm.
o There is only one type of reserve material formed by a given species; some have none at all.
o The cellular amount of these reserve materials is relatively low in actively growing cells.
 Classification based on composition:
1. Starch and glycogen
2. Poly-B-hydroxybutyric acid (PHBs)
3. Polyphosphate granules/metachromatic granules/volutin

Slide Culture Method

 It is a rapid method of preparing fungal colonies for examination and identification.

 Permits fungi to be studied virtually in situ with as little disturbance as possible.
 There is a less chance for the features that are key to identification, notably the spore-bearing
structure, to be damaged.

Morphology of a common molds:

 Aspergillus niger  Trichoderma sp.

 Rhizopus oligosporus  Mucor sp.
 Penicillum chrysogenum