11/8/2014

CULTURE MEDIUM, CULTURE

AND COLONY
• Culture medium – any material by which
microorganisms can find food and

CULTURE, CULTURE nourishment for growth and development

• Culture – ‘total growth’ or product or crop of

MEDIUM & COLONY culture medium

• Colony – ‘single growth’; result of the
reproduction of a single m.o. with the culture
medium visible by the naked eye

CULTURE
CULTURE MEDIUM Types of Culture:
• Pure – culture where the m.o. growing in the culture
medium are all of the same specie
• Mixed – 2 or more species of m.o. are growing in the
culture medium (intentionally placed)
• Contaminated – 2 or more bacterial species are growing in
the medium (accidentally placed)

COLONY

Types of Colonies:
CULTURES 1. Chromogenic – colony possessing color
2. Mucoid – colony containing mucous substances
3. Discreet- colony with same sizes and shapes
4. Surface – grows on the surface of the medium
5. Fluorescent – appears shiny
6. Chain – group of colony that are chained
7. Spreader – colony that occupies 2/3 of the total
area of a culture medium

PURE MIXED CONTAMINATED

1
 11/8/2014

Types of Culture Media:

1. Solid – AGA
Agar (2-3%) – 2 to 3 g/100 ml dist.H2O liquefy
thru
Gelatin (10-15%) – 10 to 15 g/100 ml dist. H2O heat
Albumin – solidify thru heating
TYPES OF 2. Semi-solid – where the conc. of agar is 1-1.5 %
COLONIES 3. Fluid – culture medium w/o the addition of AGA
4. Synthetic – where the exact chemical composition of the
ingredients used is known
5. Non-synthetic- where the exact chemical composition of the
ingredients used is not known
6. Differential – a series of a variety of culture media upon w/c m.o.
grow characteristically so that their isolation is facilitated

Positions for solid medium:

1. Slant
- solid culture medium in inclined
position
- intended to let the m.o. grow on the
CULTURE surface to widen the area for
MEDIA
bacterial growth
2. Butt
• solid culture medium allowed to
NON SYNTHETIC
harden in a test tube in upright
position
• intended to let the m.o. grow inside
the medium

Basic techniques needed to

study Bacteria
Observing
. Grow Bacteria
Microorganisms
• 1

• 2. Isolate Bacteria
through a Microscope • 3. Grow Bacteria in pure culture
• 4. Observe Bacteria
• 5. Identify Bacteria

2

Microscope
•Resolving Power - ability to
distinguish two distinct points
•absolute limit of the Resolving
Power is about 1/2 the
wavelength of light that is used
to illuminate the specimen
PARTS OF MICROSCOPES
• Optical or eyepiece – lens near the
observer
• Objective – lens near the specimen (LPO,
HPO, OIO)
• Arm – support the body tube and
objectives
• Coarse adjustment – bring the object into
vision
PARTS OF MICROSCOPES
• Stage – place where the slide is put
• Stage clip – holds the slide in place
• Revolving nosepiece – end piece of the
microscope body to which the objectives
are attached
• Base – supports the whole microscope
• Inclination joint – adjust the
positioning/inclining of the microscope
11/8/2014
Microscopy – Light Microscopes
Bright-field
Compound
Microscope
PARTS OF MICROSCOPES
• Fine adjustment –finer focusing of
the specimen
• Mirror – collect light from the outside
source
• Condenser – provide illuminating
cone of light to fill the aperture of
the objective.
• Diaphragm – modify the amount of
light
Microscopy – Light Microscopes
Bright-Field Microscope
Light microscope produces a dark image against brighter
background.
Commonly used to view stained cells.
Simple microscopes have single magnifying lens (like a
magnifying glass).
Compound microscopes have two sets of lenses for
magnification.
Lens closer to the eye = ocular lens (magnifying power of
10x).
Lenses closer to the object being viewed = objective lens.
(Most light microscopes used in biology have three or
four objective lenses).
3
 11/8/2014

Objective Lenses Objective Lenses

Scanning Objective Lens Low Power Objective Lens
Has yellow band around it.
Has red band around it. Magnifies objects 10x.
Total magnification = 100xTM
Magnifies objects 4x.
Start with this lens to get your
bacterial smear into crisp focus.
Total magnification = 40xTM

You will not see individual bacteria with

This lens is of no use to us in this lens, you are just using it to focus
so that you can move up to the next
looking at bacterial stains.
magnification.

Objective Lenses
High Dry Objective Lens
Oil Immersion Objective Lens
Has blue band around it. Has black and a white band
around it.

Magnifies objects 40x.

Magnifies objects 100x.

Total magnification = 400xTM Total magnification = 1000xTM

Move up to this lens after focusing Move up to this lens after focusing
your smear at 100xTM. your smear at 400xTM and
covering the 400xTM lens with a
You will not be able to clearly see finger cot.
individual bacteria with this lens. Just
get the image in focus as much as NEVER use coarse focus with high dry
possible. or oil immersion lenses!!!

Microscopy – Electron Microscopes

Two types:
TECHNIQUES IN PREPARATION FOR
Both huge, expensive
machines.
LIGHT MICROSCOPIC EXAMINATION:
Transmission Electron
Microscope (TEM):
1. Suspension in a liquid
2-D image: Transmission
Electron Micrograph • Wet Mount – place fluid containing
A transmission electron micrograph of Escherichia coli (E.coli).
organism on glass slide and cover
Scanning Electron
Microscope (SEM): with cover slip
3-D image: Scanning
Electron Micrograph • Hanging Drop – place specimen on
cover slip and invert over hollow
SEM AIDs virus attacking T4 lymphocyte
ground of slide

4
 11/8/2014

TECHNIQUES IN PREPARATION FOR Preparing smears for staining

LIGHT MICROSCOPIC EXAMINATION:
•1. Bacteria on slide
. Dried, fixed and stained smear
2

• most frequently used for observation •2. Air Dry

• advantages: •3. Bacteria are HEAT FIXED to the
• cells are made more clearly visible after
they are colored slide
• differences between cells of diff species •4. Stain is applied
and within the same species can be
demonstrated by use of appropriate
staining solutions

Bacteria are slightly negative, so are

Staining Reaction
attracted to the positive
• Stains - salts composed of a positive and chromophore of the BASIC DYE
negative ion, one of which is colored
(chromophore) •Common Basic Dyes
• Basic Dyes - chromophore is in the •crystal violet
positive ion
•methylene blue
• dye+ Cl-
•safranin
• Acid Dyes - chromophore is in the
negative ion •basic fuchsin
• Na+ dye-

Acid Dyes - used for Negative STAINING TECHNIQUES

Staining (background is stained)
1. Simple staining – coloration with
Mordant - intensifies the stain or
coats a structure to make it a single stain/color
thicker and easier to see after it is 2. Differential staining –
stained application of 2 or more stains;
Example: elicits diff between bacterial
Flagella - can not normally be seen, but a
mordant can be used to increase the diameter of
cells or parts of a bacterial cell
the flagella before it is stained
Salmonella typhosa

5
 11/8/2014

Differential Stains Gram Stain

•React differently with different •1884 Hans Christian Gram
types of bacteria •most important stain used in
Bacteriology
•2 Most Common •Divides all Bacteria into 2 groups:
•Gram Stain •Gram (+)
•Acid-Fast Stain •Gram (-)

Gram Stain Gram Stain

1. Crystal violet 2. Grams Iodine (mordant)

Gram Stain Gram Stain

3. Alcohol 4. Safranin (Counterstain)

6
 11/8/2014

SOLUTIONS GRAM POSITIVE GRAM NEGATIVE Results

APPLIED (GPB) (GNB)
Crystal violet (CV) Cells stain violet Cells stain violet

Iodine (I) CV-I complex CV-I complex formed

•Gram (+) Purple
formed w/in cells;
cells remain violet
w/in cells; cells
remain violet •Gram (-) Red
Alcohol Cell walls Lipid extracted from
dehydrated; cell walls; porosity
shrinkage of pores; increases; CV-I
permeability removed from cell •Difference - due to structure of
decreases;CV-I (COLORLESS)
complex cant pass cell wall
out of cells;cells
remain violet •Gram (+) Thick cell wall
Safranin Cells not affected, Cells take up stain,
remain VIOLET become RED
•Gram (-) Thin cell wall

Identification of a Bacteria
OTHER CHARACTERISTICS OF GNB AND GPB
Unknown
Characteristics Gram Positive Gram Negative •1. Gram Reaction
Cell wall Low in lipids High in lipids
composition (1-4%) (11-22%)
Susceptibility to
penicillin
More susceptible Less susceptible
•2. Morphology
Inhibition (basic Marked inhibition Less inhibition
dye)
Nutritional reqts Complex Simple

Resistance to More resistant Less resistant

physical disruption

Acid - Fast Stain 2 Important Pathogens

• Differential Stain - divides bacteria Mycobacterium tuberculosis

into 2 groups
• Acid - Fast
• Non Acid - Fast

• Used to identify organisms in the

Genera Mycobacterium (high lipid
and wax content in cell wall)

7
 11/8/2014

Mycobacterium leprae Acid - Fast Stain

. Carbolfuchsin (Red)
• 1

•2. Acid Alcohol

•3. Counterstain with Methylene
Blue

•Acid - Fast Cells Red

•Non Acid - Fast Blue