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MCB2010 Lab Notes
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Ex1 (P1-4) and Ex2 (P5-12)
The compound light microscope
Resolution = ability of the optics to discern 2 objects that are
Close together
Refractive index = the ability of material to bend light - light is
bent as it pass from one material into another, e. g.
air to glass. Immersion
 Oil has same reflective index as optical glass, prevent light
Bending when used between glass slide and objective lens.
Working distance = the distance between the object and the objective
lens when in focus the longer the length of the lens,
the shorter the working distance
Par focal lens = when one lens is in focus, the next lens in the
series will also be approximately in focus
Total magnification = power of ocular x power of objective lens
Tips in focusing
1.
Lower mechanical stage. Insert slide on mechanical stage, move
stage to make sure that slide moves freely without sticking.
Move scanning lens in place.
2.
While watching the stage from the side, bring stage all the way
up.
3.
While looking through the ocular, focus by turning the coarse
Adjustment knob until you obtain a sharp focus.
4.
With your eye still looking through the ocular, move the
specimen to the middle of the vision field.
5.
Switch to the next lens with a higher power.
6.
Repeat step 3, 4 and then 5.
7.
Repeat step 3, this time use the fine adjustment, then repeat
step 4.
8.
Rotate the nose plate to rest in-between the 40X and 100X lens
9. Add a drop of immersion oil, swing 100X lens in place.
10. Focus using the fine adjustment.
Focusing
Select object to focus (usually colored objects)
Focus
Center object
Change lens
Focus, center object, change lens
Prepare microscope for storage
Clean off oil from lens with lens tissue ONLY.
Put scanning lens in place.
Lower mechanical stage, center stage
Coil up electric cord.
Carry scope with both hands to storage.
Replace plastic cover.
Answer Review questions 2 and 3 b, P12 of Lab Manual
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Ex 3(P13-28)
Slide to be observed in Lab: Bacterial types, 100X objective
Identify the three common shapes of bacteria cells.
Identify the arrangement of these cells.
Common shapes of bacteria
Bacillus (bacilli), rod like cells
Coccus (cocci), oval cells
Spiral, curve cells
Coccal-bacilli, short rods
Common arrangements
Streptochains, e.g. Streptobacilli
Staphlo- bunches, e.g. Staphlococci
Diploin 2s,
e.g.
Diplococci
Photosynthetic Microbes – Includes Cyanobacteria and Algae
Slide to be observed:
1. Ocillatoria – up to 40X objective
2. Nostoc – 100X objective
Cyanobacteria
Originally thought to be more like algae, therefore, it is
also known as Blue-green algae. Recently found to be more similar to
bacteria than algae, hence renamed as Cyanobacteria (cyano=dark blue).
Distinguishable from algae by being all prokaryotes and having only
chlorophyll a. (chlorophyll = one of many photosynthetic
pigments) But without chloroplast (chloroplast = membrane bound
organelle containing chlorophyll).
Characteristics
Prokaryotes, photosynthetic
Unicellular or filamentous
Contains only chlorophyll a
No chloroplast
Primary producers
Features that can be identified in your slides:
Akinetes = found in some filamentous cyanobacteria, often enlarged with
thick walled,yellow or brown structures. Used as reproductive cells.
Heterocysts = found in filamentous forms, thick walled, light green
structures, used for nitrogen fixing.
Algae
Slide to be observed: up to 40X
1. Volvox – Observe the nucleus and presence of organelles, oval
cells forming colony
2. Spirogyra – observe the ribbon like chloroplast, nucleus,
filamentous body.
Photosynthetic eukaryotes, possess chlorophyll a + one or more
of chlorophyll b, c or d contained inside chloroplasts. Consists both
non-motile or motile species. Algae are known as primary producers.
(they form the beginning of food chains)
3 major groups of Algae
1. Non-motile, named according to pigments they contain: Green, Red
and Brown algae. Observe the following slides in the lab:
Spirogyra - green algae
Volvox - green algae
2. Pigmented flagellates
3. Diatoms - possess rigid cell wall of silica
Summary of Characteristics of Algae
Non-motile algae
pigmented flagellates
Diatomes
Motility
non-motile
flagella
gliding
Cell wall
semi rigid
thin or absent
rigid with
markings Contain
silica
Eye spot
absent
present
absent
Protozoa
Eukaryotes, include both free-living and parasitic forms, can be
divided into 4 groups based on motility. One group, Sporozoa, is
entirely parasitic and non-motile, a common example is the agent that
causes malaria. Some parasitic protozoans involve a definitive host
and an intermediate host, many are transmitted by insect vectors
(carriers).
Protista - Protozoa
4 groups based on motility:
1. Sarcodina - Amoeba, move by psuedopods.
2. Mastigophora - Euglena, move by flagella.
3. Ciliophora - Paramecium, move by cilia
4. Sporozoa - All parasites, Plasmodium(Malaria parasite), none
are motile.
Characteristics
Unicellular
Eukaryotes
No cell walls
Reproduce asexually
Include free-living and parasitic forms
Primary consumers
Slides of free living protozoa in lab
Amoeba proteus huge in size compare to the parasitic amoebae, many
Pseudopods can be identified, nucleus.
Paramecium large shoe-shaped protozoa with cilia, well defined
nucleus, mouth gullet and other vacuoles.
Fungi
Eukaryotic, unicellular(YEAST) or colony forms(MOLD). The vegetative
cell can be oval or filamentous.
MOLD
Hypha = tubular form of vegetative fungal cell, usually joint end
to end forming a long filament, can be septated (with
cross-wall in between cells) or coenocytic (no crosswall).
Mycelium = a mass of many branched hyphae, usually visible by
naked eye.
Many fungi have two morphological forms, a yeast form and a mycelial
(MOLD)form, these Fungi are called dimorphic fungi.
Consult your text book concerning the asexual and sexual
reproduction of fungi.
Slides in the lab:
Rhizopus: spores are found in a swollen structure on top
of a special stalk, the swollen sac is called
sporangium and the spores are called
sporangiospores. These spores are for asexual
reproduction.
Penicillium: spores are not enclosed in the sporangium, they
are not enclosed but are found on top of the
sporangium. These spores are called
conidiospores (meaning dust-like)
Saccharomyces: yeast, oval cells with vacuoles and granules,
reproduced asexually thru budding.
Ex 4
Blood Typing (P29-34)
Blood typing is an example of the agglutination reaction, which
Is a reaction between an antibody and an antigen.
Antigen and antibody reactions.
Antigen - soluble or particulate, mostly proteins.
Antibody-soluble, all proteins
RBC = a particulate antigen
Anti –A, anti-B, anti-D are all antibody containing reagents (sera)
Blood typing - based on agglutination reaction between a particulate
antigen (RBC) and the antibodies( anti-A, anti-B, anti Rh) specific for
that antigen.
Blood type A has
Blood type B has
Blood type O has
agglutinated
Rh + RBC has 'D'
D(anti-Rh)
'A' antigen on RBC - will be agglutinated by anti-A
'B'antigen on RBC - will be agglutinated by anti-B
no 'A' or 'B' antigens on RBC - will not be
by anti-A or anti-B
antigen on RBC - will be agglutinated by anti-
Answer Critical Thinking
questions 2 and 4, p 34
Preparation for Ex 5 (P35-40)
Ex 5 (P41-48)
Bacteria Isolation
Instruments
Inoculating loop ?for agar plates and agar slants, and broth
tubes.
Inoculating needle - for agar deeps.
Cotton swab - for agar plates.
Transfer pipettes - for broth tubes, agar pour plates.
Spreader – spread agar plates.
Media
Liquid - broth with nutrients to support growth
Solid - broth supplemented with a solidifying agent, usually agar.
Agar - extract from seaweed, no nutritious value.
Liquid at 100C Solid at 40C and below
Forms of Media
Agar plates - for growth and isolation(streak plate)
Agar slants - for growth and stock culture
Agar deeps - growth of bacteria in reduce oxygen conditions
Broth tubes - growth in liquid media
Types of medium
Simple or synthetic - chemically defined, all ingredients known,
e.g. glucose salt broth.
Complex - one or more of the ingredients are not defined chemically,
e.g. nutrient extract containing medium
Ex 6 (P55-62)
Transfer techniques
Loop sterilization - burning the wire loop or needle.
Aseptic procedures: Flaming mouth of glass containers ?to create an
Updraft to prevent aerial contamination.
To prevent contamination: Always keep cover or cap on, open only when
Needed, close ASAP when finish.
Culture techniques
Put all culture in class tray.
Tubes should be upright.
Agar culture plates should be placed lid side facing down.
Everything needs to be labeled.
Move or remove things carefully.
Growth characteristics in broth
Sediment - microbes at bottom of tube.
Flocculent - clumps of microbes in suspension.
Pellicle - microbes on top, forming a membrane.
Turbidity - microbes form a cloudy suspension.
Aseptic Transfer
A) Flame loop to red hot, cool for 1 min.
B) Remove cap, flame month of tube
C) Pick up culture, flame month of tube, replace cap.
D) repeat (B) for receiving tube.
E) Deposit culture from (C) to receiving tube.
F) Flame tube and recap, flame loop.
Answer Review questions 4 - 9; Critical thinking question 3 p 60-61
Ex 7 (P63-70)
Streaking for isolation
Mark four quadrants on the underside of a plate.
Flame loop, cool, pick up culture.
Deposit culture in sector one, flame loop, cool, streak on sector 1,
flame loop, cool.
Repeat above for sector 2.
Repeat for sectors 3 and 4 with no loop flaming.
Ex 8 (P71-74)
Bacterial Movement
Brownian movement: - Cell being moved by the vibration of water
molecule surrounding cell, no consistent directions.
Movement by flagella - Cell propelled by circular movement of
flagella, distinct direction followed by tumbling and
change of direction.
Answer Review questions 1 and 3 p73 -74 on Lab manual
Preparation for Ex9 (P75-77)
Ex 9 (P79-84)
Bacterial Stain
Stain is used to increase the contrast between cells and background.
Two chemical groups in a stain
Chromophore - imparts color to the stain.
Auxochrome - imparts chemical properties such as electrical charges
to stain.
Bacterial Stains
Basic stains - carry a positive charge, usual stains for bacterial
cells, which carry negative charges on cell wall.
Acidic stains - carry a negative charge, generally used for
background staining.
Smear Preparation
1. Spread or create a suspension of bacterial cells on a clean glass
slide(dime size).
2. Air dry thoroughly. Ideal thickness ?ability to see printed words
through the dried smear.
3. Heat fix by passing several times on top of flame.
Simple Stain – using one stain only
Cover smear with stain for 1 min.
Tilt to drain stain from slide.
Rinse thoroughly with water.
Blot dry and examine.
Answer review questions 3 ans 4 P84
Ex 10 (P85-90)
Gram Stain
Primary Stain - Stain with Crystal Violet for 1 min.
Tilt to remove stain, rinse with water.
Mordant - Add Gram’s iodine, stain for 1 min.
Tilt to remove solution, rinse thoroughly.
Decolorization - Tilt slide, drip Ethyl alcohol over smear to
decolorize smear. Observe the lower edge of the smear
carefully, stop alcohol decolonization as soon as the
run off from the smear is clear.
Rinse with water, shake off excess.
Counter Stain - Add safranin, stain for 50 seconds.
Tilt to remove stain, rinse thoroughly.
Blot dry.
Answer All revuew questions p89-90
Ex 11 (P91-96)
Acid-fast stain:
A differential stain which distinguish acid fast
organisms from Non-acid-fast organisms, based on
the presence of Mycolic acid, a waxy substance in
the cell wall of acid-fast organisms. The primary
stain is driven through the cell wall by heat. An
example of acid-fast organism is:
Mycobacterium sps.
Ziehl-Neelsen acid-fast stain includes the following steps:
1.
Preparation of bacterial smear
2.
Air drying of smear
3.
Heat fixing of smear
4.
Primary stain - Carbo-fushin
5.
6.
7.
8.
9.
water rinse
Decolourisation - acid alcohol
water rinse
counter stain - Mythylene blue
water rinse and blot dry
Answer Review questions 2, 3, Critical Thinking 1 and 3
Ex 12A (P97-98)
Endospore stain:
A special stain which stains endospores, commonly
present in species of Bacillus and Clostridium.
Steps in performing the Schaeffer-Fulton Endospore stain
1.
Prepare bacterial smear
2.
Air dry smear
3.
Heat fix smear
4.
Primary stain - Malachite green with heat on a steam bath
5.
Water rinse and decolonization
6.
Counter stain with Safranin O
7.
water rinse and blot dry
Ex 12B (98-103)
Capsule stain: A special stain which reveal the polysaccharide capsule
of some bacteria. Capsular material is soluble in water,
copper sulfate is used to wash bacteria smear isnstead.
Steps in performing the capsule stain (direct staining using basic
Stain)
*DO NOT PREPARE SMEAR THE REGULAR WAY; NO HEAT FIXING IS NECESSARY
1.
2.
3.
4.
Add 1 drops of crystal violet on a clean slide.
Aseptically add loopful of culture and mix with the crystal
violet.
Prepare a thin film smear as directed.
Air dry and examine with oil immersion lens.
Answer review questions 1, 2, 3b, 3c, 4, 5 on page 102-103
Ex 13 (P105-108)
Negative Staining:
Staining the background instead of the bacterial
cells by use of an acidic stain, cells and
associated structures such as capsule will appear
as empty objects on a color background.
Steps in performing a negative stain
*DO NOT PREPARE SMEAR THE REGULAR WAY; NO HEAT FIXING IS NECESSARY
1. Add a drop of Nigrosin to one end of a clean slide.
2. Aseptically transfer a loopful of bacteria and mix well with
stain.
3. Prepare and thin film smear as directed.
4. Air dry and examine with oil immersion lens.
Answer all review questions
Introduction to Ex 15 (p113-p115)
Ex 15
Enumeration of bacteria growth curve (p117 – 118)
Bacterial growth curve
typically, consists of 4 phases:
1. Lag phase - no increase in cell number, bacterial is
preparing for cell division by duplication of DNA
and production of various materials needed for
cell multiplication.
2. Log growth phase - more cells are being born than die,
resulting in greatest increase in cell number per
time interval.
3. Stationary phase - nutrients are getting used up by
the rapidly dividing cells, resulting in equal
number between new cells generated and those that
died.
4. Log death phase - continue depletion of nutrients,
more cells are dying than being born.
Exercise modifications: Procedure
1. Obtain 6 tubes of BHI broth, label as 0, 30, 60, 90, 120, 150
min.
2. To each of the above labeled tube, add 0.1 or 2 drops of E. coli
stock culture, mix well.
3. Put all tubes except the 0 min in 37 C incubator.
4. Transfer the content of the 0 min tube to a measuring tube.
Measure and record the Optical Density of the content in tube
0.
5. Transfer content back to the culture tube, discard both tubes.
6. At the end of each time interval as marked on each tube, repeat
Step 4 and 5 above.
7. Plot graph using the data obtained.
Spectrophotometer count
Light Transmission in spectrophotometer reading - Bacterial
concentration is indirectly proportional to amount of
light transmitted.
Optical density in spectrophotometer reading - Bacterial
concentration is directly proportional to amount of light
absorbed.
Requirements before reading sample:
Blank (zero) reading necessary with the same medium as the
culture to be measured.
Standard curve showing relationships between
absolute bacterial number an spectrophotometer
readings had to be constructed.
Answer review questions 1 & 4 on page 122
Introduction to Ex 16, 17 (p127-p129)
Ex 16 A(p131 – 134)
Antimicrobial agents
Antimicrobial agents includes:
a) antibiotics, which are produced by living organisms,
usually microorganisms, to inhibit growth of others.
b) antiseptics and disinfectants, which are often chemical
compounds made by man to inhibit growth of microbes.
Test for susceptibility of micro-organisms towards any agents
involve growing Of micro-organisms in the presence of the substance to
be tested. The one that you are going to do in the lab is the disc
diffusion test where antimicrobial agent in a paper disc is allowed to
diffuse into the medium which had been previously seeded with a lawn of
bacteria for the test. The size of the area where there is no bacterial
growth indicated the zone of inhibition. The zone of inhibition is
measured in mm and represents the diameter of the clear circle where
there is no growth of bacteria.
The Kirby-Bauer antibiotic sensitivity test depends on the
ability of antibiotics diffusion into surrounding medium. A
standardised plate, Mueller-Hinton plate, is used to limit various
physical factors that may affect the diffusion rate.
MIC = minimum inhibitory concentration, which is the minimum
concentration of antimicrobial agent needed to inhibit the growth of a
bacterium.
The MIC is different for each bacteria and varies with
different antimicrobial agent. The effectiveness of your laboratory
test should be compared to a standard chart to see if it falls within
the effective range.
Answer review questions 3 page 148
Ex 17 B (141-146)
Exercise Modification:
Material: 5 mm filter paper discs
Solutions of various disinfectants and antiseptics
Procedure:
1. Obtain a Mueller-Hinton agar plate, mark off the appropriate
number of sectors, one for each solution to be tested, label.
2. Using forceps, Dip and saturate a paper disc with
disinfectant solution, drain excess solution by touching
the side of the container.
3. Carefully lay down the disc onto the middle of the labeled
Sector, tap disc gently to ensure contact with agar.
4. Repeat the above until you finish with all the solutions to
be tested, secure lid and incubate till next lab.
Ex 18
Temperature as a means of control of microbial growth. Refer to
your lab manual,
P149,
Growth Temperatures
Bacteria can be separated into several group based on their growth
Temperature range:
Psychrophiles
Mesophiles
Thermophiles
0 - 200 C
25 - 400 C
45 - 650 C
Types of Heat for Control of microbial growth:
Dry Heat- not efficient, requires long exposures, for glass wares and
solids.
Moist Heat- efficient, various types:
Autoclave - steam under pressure, for sterilization, 15 PSI,
1210C, 15 minutes.
Boiling - not effective for endospores.
Pasteurization - prevent spoilage, 630C for 30 minutes.
Answer questions 2, 5, 6
p 152.
Ex 19
See lab manual p155 for theory
Answer questions 1, 2 and Critical thinking 1, 2 p160
Radiation Control of Microbial Growth, two major forms of radiation
that can affect the growth of micro-organisms:
Ionizing Radiation - High energy, high penetration, generates
free electrons and free radicals, damage
DNA molecules. Example: X rays, Gamma
rays.
Non-ionizing Radiation - Low energy, low penetration.
Example: UV light, generates
thymine dimers, generates mutations in
DNA, reversible through repair
mechanisms which operate in darkness.
Ex 21
See lab manual p169 for principle
Direct Microscopic Count
Hemocytometer - fixed volume counting chamber, count 5 squares in
center grid.
Calculation average of 5 squares x 25 x DF
DF= dilution factor
Ex 22
Water Analysis
Fecal contamination of water
Indicator: E. coli
3 Basic tests
presumptive - lactose fermentation with gas production
confirmed - growth of organism on selective and differential
media (EMB agar)
completed - gram stain and growth on lactose broth with acid
and gas production
Answer Review Questions
1, 4 page 182
Ex 23
Sugar Fermentation
Uses glycolysis to generate energy – Glucose Broken down to form
Pyruvic acid.
Pyruvic acid is the intermediate, it is further broken down either
in:
Alcohol fermentation - end product is alcohol and gaseous CO2
Homolactic fermentation - end product is lactic acid
Heterolactic fermentation - end products are mix acids,
alcohol, gas
Sugar Fermentation Broth Tube
Contents:
Nutrient Broth - supports proteolysis(peptonization)for nonfermenters, produce basic end product.
Sugar - fermentation produces acids with or without gas,
acid decreases pH.
Phenol red - acid base indicator, yellow = acid
red = base
Durham tube - inverted tube for gas collection.
Answer review questions 1,4, 5 page 191 -192
Ex 24
24A
Microbial Exoenzymes
Carbohydrate Hydrolysis
Starch = complex carbohydrate
Building blocks = simple sugar (glucose)
Hydrolytic Enzyme Amylase breaks down starch to glucose.
Test for enzyme activity = presence or absence of starch.
Iodine + starch = Blue complex
Microbial Exoenzymes
Gelatin Hydrolysis
Building blocks = amino acids.
Hydrolytic Enzyme gelatinase breaks down gelatin to amino acids.
Test for enzyme activity - lose of ability of gelatin to gel at
room or temperature.
24B
Microbial Exoenzymes
Lipid Hydrolysis
Building blocks = glycerol (an alcohol) and fatty acids.
Hydrolytic Enzyme lipase breaks down lipid to fatty acids and
glycerol.
Test for enzyme activity(lipolysis)
presence of lipid = opaque agar
absence of lipid = clear agar.
Microbial Exoenzymes
Casein(milk protein) Hydrolysis
Building blocks = amino acids.
Protease Enzyme breaks down casein to amino acids.
Test for enzyme activity - proteolysis
presence of proteolysis = clear zone
absence of proteolysis = opaque agar
Ex 25
Nitrate Reduction
NO3
Red
Nitrate reagent A + B
----------------------------
Nitrate reagent A + B
NO3 ---------------------------colorless
NO2
NO2 ------
NH3 --------
N2
Nitrate reagent A + B
NO3 -------------------------------------------------colorless
NO3
Red
Nitrate reagent A + B
----------------------------
NO2
Manual reduction
using zinc
Answer review questions 3, 4 page 202
Ex 26
IMViC Test
I = Indole production from oxidation of amino acid Tryptophan
SIM agar deeps
1 semi-solid agar for motility
motile bacteria – growth throughout agar
non-motile bacteria – growth along stab
2 Tryptophan for Indole production
Presence of Indole Turns Kovacs reagent red
3 Test for Hydrogen Sulfide production
Black precipitate of Ferric sulfide in agar
Methyl Red Test
Test for the product of Glucose oxidation
Stable acid – turns MR reagent to red
Example – E. coli
Acetoin – non acidic, turns MR reagent yellow.
Example – E. aerogenes
VP Test
Voges Proskkauer test
Test Product of glucose oxidation
Neutral products – turns Barritt’s reagent pink.
Example: E. aerogenes
Citrate Test
Test for the fermentation of citrate as a sole source of
carbon.
Positive results:
1. Growth and/or
2. Alkaline product turns Bromthymol Blue from green to
blue.
Ex 27
Catalase Test
Results of survival in oxygen
Toxic products - H2O2 and superoxide
Enzymes needed to detoxification
Catalase – converts H2O2 to water and oxygen.
Superoxide dismutase – converts superoxides to H2O2.
Use of Selective or Differential Media
Selective and Differential Media
Mannitol Salt agar
Salt selects for halophile
Mannitol differentiates Mannitol fermenting from non-fermenting
organisms.
EMB agar
dyes inhibits Gm +, selects for Gm Lactose differentiate fermentation + vs – ve organisms.
Differentiate heavy vs light acid producers.
Also
MacConkey agar – very similar to EMB
Crystal violet dye inhibits Gm +, selects for Gm -.
Lactose differentiates fermenting from non-fermenting organisms.
Differential Media
Blood Agar – test for hemolysis of RBC
Alpha – incomplete hemolysis, release and partial degradation
of hemoglobin, red or green zone.
Beta – complete hemolysis, total degradation of released
hemoglobin, clear zone.
Gamma – no hemolysis, absences of any zones.
Selective Media
Phenylethyl Alcohol Agar
Select for Gm +, inhibits Gm - organisms.
Generally used for isolation of Staphylococcus and
Streptococcus from mixture.
Rapid Identification Test
Enterotube
Commercially produced
rapid test for the identification of Enterobacteriaceae
Advantages – fast results, less labor intensive, provides
indications for early treatments.
Answer questions
p 212
Question 6;
p. 216
#2 and 3;
p. 222 # 1 and 2
Good Luck, you have make it this far.
2 more hurdles to get thru and you
will finish.