Micro Biology Lab Packet

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Microbiology Lab Packet #2

Think of these lab packets as homework for the lab part of the class. They are designed to help prepare you for the departmental lab exams. To answer these questions you will need to use both the lab workbook as well as your text book. In addition to these questions, you will need to turn in the signed data sheets from your workbook.

19. Describe the molecular basis behind Gram staining. What part of the bacteria is stained with the primary stain, what part is stained with the counter stain? Why do we wash alcohol over the slide before the counter stain?

18. Describe in great detail the steps in performing a Gram stain. Include incubation times. Deﬁne primary stain and counter stain. In the Gram stain, what dye is primary stain, which is the counter stain?

Special Stains (Capsule Ex. 3.9, Endospore Ex. 3-10, Flagella Ex. 3.12)

21. What is the stain is shown in the picture below? What is stained green, what is stained red?

22. Describe in great detail the steps in performing an endospore stain. Include incubation times.

20. What does it mean to be Gram positive, Gram negative?

23. Label the different ﬂagellum arrangements seen below.

24. Describe the negative staining technique and determine when it is used.

Colony Morphology Ex 2.2

25. We can learn some information about bacteria by just looking at its growth on an agar plate. What is a small isolated growth of bacteria on an agar plate called and what can it tell us about the bacteria?

Micro lab packet 2

Kirby-Bauer Method Ex 7.3

26. Describe in great detail the technique used to determine bacteria sensitivity to antibiotics?

27. What is the type of media used in the Kirby-Bauer test and why is it used?

Describe what is observed for a bacteria that is sensitive to antibiotics in a Kirby-Bauer test, and to a bacteria that is not sensitive to antibiotics.

28. How does the antibiotic get from the disk into the agar?

Does the agar have an antibiotic beyond the zone of inhibition?

29. Deﬁne the MIC and explain how it is used to determine if a bacteria is resistant or sensitive to the antibiotic.

Is the presence of a clear zone around the disc enough evidence to determine antibiotic resistance?

Micro lab packet 2

Disinfectants Ex 2.14

30. Compare your results with the class data.

Which germicide was most effective and at what concentration?

Which was the least effective? Defend your choices.

31. Which organism seemed to be most resistant to the germicides?

32. What purpose did the four controls serve? Be speciﬁc.

Include Completed Data sheets for:

1. Data Sheet 3-7 (pages 201-202) Sketch & id whether Gram + or Gram – and Questions 1-2

2. Data Sheet 3-9 (pages 213-214) Sketch capsuled stained bacteria; Questions 1 & 2

3. Data Sheet 3-10 (pages 219-220) Sketch Endospore Stained bacteria; Questions 1-4

4. Data Sheet 3-12 (pages 227-228) Sketch Flagella Stained bacteria; Questions 1 & 3

5. Data Sheet 2-14 (pages 139-140) Questions 1-3

6. Data Sheet 7-3 (pages 535-536) Questions 1-7

Gram Staining 3.7

33. What is the difference between disinfectants and antiseptics?

Lab 1
Aseptic Transfers & Inoculation Methods (Ex 1-3)
Streak Plate Method (Ex 1-4)

Aseptic Technique
Aseptic = free of contamination
Purpose:
To keep our cultures from becoming contaminated
To keep the lab environment from becoming contaminated
To keep us from becoming infected by our cultures

Labeling cultures
Always include:
The name of the organism or specimen
Your name or initials
The date inoculated
For broths/slants, label so you can still see growth
For plates, label on the bottom, the side with the growth medium
Label before you inoculate

Sources of Contamination
From our culture’s point of view:
us
the air
objects & surfaces in the environment

From our point of view:
the culture

Materials we’ll be using today
Culture Media
Broths (1 per student)
Slants (1 per student)
Plates (2 per student)
Inoculating Loop
Bunsen Burner
Flint Striker

Demonstration of slant to broth transfer
(See Ex1-3, p. 32-37 in lab manual)
Tube of broth & labeling
Aseptic technique
Inoculum
Transfer to fresh broth
Incubation at 37°C

Demonstration of slant to slant transfer
Slant & labeling
Aseptic technique
Inoculum
Transfer to fresh slant
Incubation at 37°C

Streak Plate Method
Purpose: To check or establish the purity of a culture
Pure culture = contains a single species
Mixed culture = contains more than 1 species
Principle: Organisms are diluted out over the surface of a growth medium, so individual cells will grow to form visible colonies
Colony = a visible mass of cells
1 cell = 1 colony-forming unit (CFU);
The assumption is that 1 cell grows to form 1 colony

Streak Plate Method (see p. 43)
Quadrant Streak Method
* FLAME    
Heat loop
Inoculate culture
Streak
1st quadrant

A good streak plate
Is appropriately labeled
Has appropriate pattern for streak lines
Uses the entire plate
Has well-isolated colonies
Is free of contamination
One colony type

Demonstration of Streak Plate Method
Agar plate & labeling
Aseptic technique
Inoculum
Streak pattern
Incubation (inverted) at 37°C

For your review:

Video of aseptic technique (2 min)

Video of streak plate method (1½ min)

Expectations: Work as individuals
Aseptically transfer from stock slant culture to a fresh broth and to a fresh slant – use “S. marcescens ” or “M. luteus” or “E. coli” or “S. sapro”
Label appropriately with the organism, your name or initials, & date
Aseptically Streak 1 plate using the quadrant method
Use “culture used above”
Label appropriately
Incubate in plate in inverted position
Aseptically Streak a 2nd plate using the quadrant method
Use “mixed culture”
Label appropriately
Incubate in plate in inverted position
Put all your cultures into the incubator (37°C), on the shelf for this section

Expectations: Work as individuals
After inoculations are complete, Work on Simple Stain from last week
Ex. 3.5, pg. 186-187 Making a Bacterial Smear and Simple Stain procedure.

Gather around for demonstration
(use real inoculum)
Broth to broth transfer
Plate to slant transfer
Streak plate method

Disinfectants & Antiseptics
Antibacterial Susceptibility Test
Ex 2-14 Disinfectants & Antiseptics
Ex 7-3 Antimicrobial Susceptibility Test

Chemical Agents – General Principles
Sterilization
– killing or removal of all life forms
Disinfection/Antisepsis – removal of pathogens; reduction in the number of organisms, so they pose no danger of disease
Disinfectant – chemical agent used on an inanimate object

Antiseptic – chemical agent used externally or topically on living tissue

Effect of Chemical Agents on Bacteria
There are many different methods to assess effects of chemical agents on bacteria
Laboratory methods – Assess the properties of agents under laboratory conditions
E.g., methods to evaluate antibacterial potency
E.g., methods to evaluate –cidal or –static activity
In-use methods – Assess the effectiveness of agents under conditions similar to actual use
E.g., methods to evaluate performance for particular uses or in real-life situations
E.g., methods to compare performance of 2 agents

Chemical Agents – Tube Dilution Procedure
Determining the antibacterial potency of an agent against Staphylococcus aureus (S. aureus)
or Escherichia coli (E. coli)
Agents: Isopropanol, H2O2, Bleach, or Lysol
at various concentrations
Inoculated with S. aureus or E. coli by swab technique with fresh broth culture

Determine the lowest concentration of that agent that inhibits growth of the test organism

1.
4.
2.
3.
5.
6.
8.
7.
1 min.
10 min.
dry for 10 min.

Effect of Chemical Agents
So which procedure is a laboratory method?
Beads procedure
Which procedure is an “in-use” method?
Fingertip antisepsis procedure

Chemical Agents
Active ingredient should be noted on the container
Class = the general group to which the active ingredient belongs:
Product Class
Isopropanol Alcohols
Hydrogen Peroxide Oxidizer
(H2O2)
Bleach Oxidizer (Sodium Hypochlorite)
Lysol Quaternary Ammonium
Compounds

Effectiveness
(Lowest Concentration of Reagent Needed)
Organism Bleach H2O2 Isopropanol Lysol
S. aureus
E. coli

Antibacterial Susceptibility
Ex 7-3 Antimicrobial Susceptibility Test

Why Study Antibacterial Susceptibility Testing?
Clinical labs routinely perform “culture & sensitivity” testing
Ideally, effective antibiotic treatment is directed against the specific pathogen infecting the patient
Some strains of a species have acquired resistance to antibacterial agents

Antibacterial Agents
Spectrum of activity
Broad spectrum – Active against a broad array of bacteria, including both Gram(+) & Gram(-) organisms
Narrow spectrum – Active against a limited range of organisms

The Sites of Activity in a Bacterial Cell for Various Antibiotics
Chloramphenicol inhibit protein synthesis
Ciprofloxacin: Iinhibit DNA gyrase
Trimethoprim: inhibits bacterial DNA synthesis
Penicillin: inhibit the formation of
peptidoglycan cross-links in the bacterial
cell wall

Kirby-Bauer method for determining antibacterial susceptibility
Disc-diffusion method

Antibiotic discs
Contain defined amount of a specific antimicrobial agent
Agent diffuses out of disc radially, into the medium
Forms a concentration gradient in the medium

Zone of inhibition
Zone size determined by concentration of the agent that inhibits bacterial growth

Assessing Antibacterial Activity
How to get from a zone of inhibition to effectiveness (or lack of effectiveness)?

Measure zone of inhibition
Bigger zone is better
Zone size alone doesn’t determine susceptibility or resistance

Must consider
Factors associated with the test method
Concentration of drug in the body after a usual dose

Kirby-Bauer method for determining antibacterial susceptibility
(Mueller-Hinton Agar used)
non-selective, non-differential medium (almost all organisms plated will grow)
has a few properties that make it excellent for antibiotic use
contains starch. Starch is known to absorb toxins released from bacteria, so that they cannot interfere with the antibiotics.
it is a loose agar, which allows for better diffusion of the antibiotics than most other plates. A better diffusion leads to a truer zone of inhibition.

Interpretive Criteria
Interpretive criteria convert zone size (mm) to 1 of the following categories:
S = susceptible; pathogen with that zone size will likely be killed/inhibited by the concentration of drug in the bloodstream provided by the usual dose of that agent
This agent should provide effective therapy
R = resistant; pathogen with that zone size will NOT likely be killed/inhibited …
Select a different agent for therapy
I = intermediate; provides a buffer zone to prevent small differences in the test from having a major impact on interpretation
Select a different agent for therapy

Antibacterial Susceptibility – Day 2
Measure diameter of zone of inhibition with ruler (in mm)
Record data for all 4 agents
& 2 different organisms (Worksheet 7-3 pg. 661)

Use interpretive criteria to convert zone size (mm) to a category (page 271):
S = susceptible
I = intermediate
R = resistant

Zone diameter (mm)

Interpretive Criteria
Convert zone size (mm) to S, I, or R

Agent Zone size (mm)
Susceptible
Intermediate Resistant
Chloramphenicol – C30 Enterobacteriaceae & staphylococcus
>18
13 – 17
<12 Ciprofloxacin - CIP Enterobacteriaceae & staphylococcus >21 16 – 20 <15 Trimethoprim – TMP5 Enterobacteriaceae & staphylococcus >16 11 – 15 <10 Penicillin – P10 Staphylococcus >29
<28 Expectations for Today: Work as table-teams 2 Mueller-Hinton Agar plates & 1 pkg swabs per table Inoculate each plate with 1 organism: Swab in 3 directions for confluent growth Staphylococcus aureus Escherichia coli Apply 4 different discs to each plate Chloramphenicol - C30, Ciprofloxacin – CIP, Trimethoprim – TMP5, Penicillin – P10 Use flamed forceps; tap disc gently for good contact with agar Incubate in the inverted position at 37°C Disc will not fall off, even upside down in the plate We will observe/interpret results next lab C30 CIP TMP5 P10 Disinfectants & Antiseptics Antibacterial Susceptibility Test Day 2: Interpretation of Results Ex 2-14 Disinfectants & Antiseptics Ex 7-3 Antimicrobial Susceptibility Test Expectations for Today Read & interpret antibacterial susceptibility test Measure zone size (mm) Use interpretive criteria to convert zone size to S,I,R Summary of Class Data Agent S. aureus E. coli Chloramphenicol - C30 Table 1 = Table 2 = Table 3 = Table 4 = Table 1 = Table 2 = Table 3 = Table 4 = Ciprofloxacin - CIP Table 1 = Table 2 = Table 3 = Table 4 = Table 1 = Table 2 = Table 3 = Table 4 = Trimethoprim – TMP5 Table 1 = Table 2 = Table 3 = Table 4 = Table 1 = Table 2 = Table 3 = Table 4 = Penicillin – P10 Table 1 = Table 2 = Table 3 = Table 4 = Table 1 = Table 2 = Table 3 = Table 4 = Ex 3.9 Capsule Stain Ex 3.10 Endospore Stain Ex 3.12 Flagella Stain Week 3 Today we’re doing: Differential stains on some reference organisms: Capsule stain Endospore stain Flagella stain Finish Gram Staining Differential Stains: Endospore Stain Differentiates bacteria based on ability to form endospores or not Can often visualize the presence, shape, and location of spores within a vegetative cell Spores are resistant forms; heat is needed to drive the stain into the spores Spore-forming bacteria include Bacillus species and Clostridium species The Genus Clostridium Gram-positive, spore-forming rods Anaerobes > 100 species
Pathogens:
C. tetani – tetanus
C. botulinum – botulism
C. perfringens – gas gangrene
C. difficile – antibiotic-associated colitis
Nonpathogens:
Live in large bowel as commensals
Clostridium tetani
Clostridium difficile

Spore stain (p 217)
Endospores are resistant; stain does not readily penetrate spores
Spores will stain with Malachite Green (primary stain) and heat
Decolorize with water; vegetative cells will lose the malachite green
Counterstain with safranin
Spores will appear _________ and vegetative cells will appear ________.
blue-green
red/pink

Spore
Stain

Spores will appear _________ and vegetative cells will appear ________.
blue-green
red/pink

The Genus Bacillus
Gram-positive,
spore-forming rods
Aerobes
 40 species
Only pathogens:
B. anthracis – anthrax
B. cereus – food poisoning
Nonpathogens:
B. subtilis – source of enzymes
B. polymyxa – source of antibiotics
Bacillus anthracis

Spore Stain – think about it
When do spore-forming bacteria undergo sporulation?
Are spores likely to be observed in fresh cultures or on a rich medium?
If spores are not observed:
it might mean that spores weren’t produced under the conditions of growth
or, it might mean this organism is not a spore-former

Expectations – Work as individuals
Spore stain – p. 217
Prepare a smear on a slide each for:
Bacillus subtilis and Bacillus cereus
Observe slides using 100X oil immersion objective
Observe Flagella stained slides on instructors table at front

After that, continue with your prepared simple and Gram stained slides

p. 217
Steam w/ Malachite Green stain for 10-15 min

Differential Stains: Capsule Stain
Differentiates bacteria based on ability to produce a large capsule or very little/no capsule
Capsule is one kind of glycocalyx
Usually composed of polysaccharide
Protects cell from drying and from host defenses (anti-phagocytic)
Caution: Capsules are somewhat fragile:
Destroyed by heat-fixing
Can be washed away
Staining for a capsule is useful for identification and classification

Capsule stain (p 212)
Small amount of bacteria is suspended in Congo red – an acidic dye (Ignore the serum)
Acidic dye will stain/not stain the bacterial cell
Draw out the drop to create a smear
Do not heat fix your smear! Let it air dry
Flood the smear with Maneval’s stain
Basic dye will stain/not stain the bacterial cell
Gently rinse with water.
Observe using oil immersion lens (100X)
Distinguish between a tiny halo (from cell shrinkage) and a true capsule.

(p 212)

Capsule
Stain

Flagella
Flagella allow for motility
Bacterial flagella rotate rather than whip
Arrangements
Monotrichous
Peritrichous
Lophotrichous
Amphitrichous
Presence/absence, number & placement of flagella are useful for identification & classification

Monotrichous
Amphitrichous
Lophotrichous
Peritrichous
A
B
C
D

How would you determine if a bacterium has flagella?
Look for flagella microscopically
But hard to see with the light microscope
Look for motility
an indirect assessment of the presence of flagella
So, if a cell is motile, it must have flagella
We’ll look for motility using a wet mount

Bacterial Motility
Videoclip2 of Bacterial Motility

Videoclip1 of Different Types of Bacterial Motility

Motility Test Medium
A semisolid medium, containing a reduced amount of agar
Inoculate vertically, using an inoculating needle
Motility is apparent if organisms grow and spread away from the line of inoculation
Nonmotile organisms will only grow at the line of inoculation

Motility Test Medium
+ – – +

Welcome to General Microbiology Lab
Biol 2421

Introductions
My name is Professor Troy Giambernardi
Pronounced “Gim-burr-nardi”
Please introduce yourself to the students at your table.
Name, program of study, how long you’ve been a student at Lone Star College, how & how far you come to get here
Class list: Please tell me your name, using the name you would like to be called
Some information, please: Student Information Sheet
Next lab, I’ll circulate a seating chart

Syllabus and Schedule are posted on Desire 2 Learn (D2L)
Class & Lab will use the same D2L site
Lab Syllabus
Lab Schedule

Safety Considerations
Respect live microorganisms!
Please let me know later if you are immunocompromised or immunosuppressed
No food or drink in the laboratory
Lab coat, smock, or old shirt is highly recommended
Close-toed shoes are highly recommended
You are responsible for your own Gloves
Must cover any cuts/open sores with gloves or band-aid
Other safety items
Raise any safety concerns or questions

Procedure for Disinfecting a Lab Spill
Cover generously with Lysol®
Lay paper towels on top
Let sit for 5-10 minutes
Clean it up
Wear gloves and/or use brush & dustpan
Be cautious of broken glass
Discard into biohazard trash

Basic Rules & Procedures for Working in the Biology Laboratory
Please sign Basic Rules and Procedures Sheet circulating around the lab
Put on the instructors table up front
A duplicate copy is at the back of the class syllabus on D2L

Before you come to lab
Please look at scheduled activities on the lab schedule in the syllabus
Please read the lab exercises in the Lab Manual
Microbiology Laboratory Theory & Application, 4th edition; Leboffe & Pierce
Please bring the lab manual with you to lab.

Summary of how lab runs:
Put away gum, drinks, cell phones, etc.
Disinfect bench with Lysol.
Listen to short introduction – take notes.
Do the lab exercise(s).
Complete “Data Sheet” questions at the back of each section of your lab manual germane to the Exercise(s) we are working on.
These will be collected along with the posted Lab Packet questions for grading.
Disinfect lab bench.
Wash hands before you leave.

Exercise 1-1
Glo Germ Hand Wash

Glo Germ Hand Washing
Purpose:
To assess proper hand washing techniques
To keep the lab environment from becoming contaminated
To keep us from becoming infected by our cultures
Follow protocol from instructor

Glo Germ Demo

Bathroom Hygiene Myth | MythBusters

Toilet Germs Reach Your Tootbrush?

Inoculating Petri Plates from Environment Cultures
Step 1: Obtain a sterile cotton swab, sterile saline, and one agar plate.
Step 2: Choose a surface to sample.
Step 3: Label the bottom of your plate with sample name, your name or initials, and date.
Step 4: Remove sterile swab, wet swab with saline, and sample surface of interest with wetted swab.

Inoculating Petri Plates
Step 5: Holding the petri dish lid at an 30°-45° angle, work the contaminated swab from the outside of the plate toward the center in a zig-zag pattern that covers approximately 25% of the plate surface (think pie or pizza slice!).

Inoculating Petri Plates
Step 6: Turn the petri plate 90° to the right, dragging the inoculation loop through the last section of the plate, moving from the outside to the inside in a zig-zag motion.
Step 7: Repeat this process twice more until the entire plate surface is covered.
NOTE: If you are trying to isolate individual colonies, each turn of the dish will give you fewer microbes so that you can distinguish individual colonies.

If you streak your plates correctly, the pattern should look like this on your plate.

*
If you streak your plates correctly, the pattern should look like this on your plate.

Inoculating Petri Plates
Step 8: Replace plate lid and invert the plate for incubation at 37°C.
Plates are inverted (upside down) to avoid condensation spilling onto your cultures.

Lab 2
Ex 3-1 Introduction to Microscopy
Ex 3-5 Simple Stains

Housekeeping Items
Did you Lysol your benchtop yet?
My name is Professor Giambernardi
Pronounced “Gim-burr-nardi”
Please introduce yourselves at your tables
Seating chart & attendance
Is everyone finding things on D2L?
Remember to purchase sharpies & gloves

Today we will do 3 things:
Observe our cultures inoculated last lab
Become familiar with our microscopes
Perform some simple stains?

Let’s check our cultures from last lab
Nutrient agar plates
Did they grow? How can you tell?
Did you see a difference between unwashed and hand sanitizer?

When finished, discard cultures in biohazard trash.
Remember to answer questions in “data sheets” (back of lab section) germane to the sections we are working on

Microscopy
Light microscope – uses visible light as the energy source

Brightfield microscopy
Produces an image made from light that passes through a specimen
The background appears bright
Objects appear darker, or might lack contrast

Microscopy
Compound microscope – has two lenses that magnify the image
Ocular
Objectives

The Compound Light Microscope
Light source at bottom
3 lenses produce an image:
The condenser lens focuses & concentrates light to evenly illuminate the specimen
The objective lens collects light, magnifies, and forms an image that you see with your eyes.
The ocular lens further magnifies the image formed by the objective lens.

Concept #1: Magnification
Magnification = the size of the image
Four different objective lenses:
scanning low power high power oil immersion
4X 10X 40X 100X

Ocular lens: 10X
Total magnification = (objective mag) x (ocular mag)
Parfocal = object in focus under 1 objective will be in focus under all objectives
Paracentered = each objective will successively focus on the center of the previous field of view

Magnification
Field diameter = the actual diameter of the area viewed, using those particular lenses
Units will be a distance (mm, µm)
As magnification increases, field diameter decreases
Objective Field diameter
scanning 4.5 mm
low power 1.8 mm
high power 0.45 mm
oil immersion 0.18 mm

Magnification
Depth of Focus = the amount of vertical space that appears in focus, using those particular lenses
Similar to depth of field in photography

As magnification increases, depth of focus _____?______
decreases

Concept #2: Resolution
Resolution – the clarity of the image
Resolution & Car Headlights

UNRESOLVED PARTIALLY RESOLVED RESOLVED

Concept #2: Resolution
Limit of resolution (resolving power)
How far apart 2 points must be to be distinguished as separate
Indicates the size of the smallest object that can be clearly observed with those lenses
The limit of resolution is decreased as wavelength is decreased.
High magnification without high resolution is “empty.”

Magnification & Resolution

Limit of Resolution
As magnification increases, the limit of resolution gets smaller

For example,
Eye 0.1 mm (millimeters)
Light Microscope 0.2 m (micrometers)
Scanning EM 3.0 nm (nanometers)
Transmission EM 0.2 nm (nanometers)

Question about Resolution:
We just said the limit of resolution (resolving power) of our light microscopes is 0.2 m

We want to see a bacterium that has a diameter of 1 m

Will we be able to see this bacterium using our microscope?
Why or why not?

Concept #3: Working Distance
The amount of clearance between the slide and the bottom of the objective lens
As magnification increases, working distance _________
Don’t crash your objective lens into the slide!
decreases

Concept #3: Working Distance
The amount of clearance between the slide and the bottom of the objective lens

4X
40X
10X

Concept #4: Refraction
Refraction – bending of light as it passes through an object
Refractive Index – a measure of the light-bending ability of a medium

Concept #4: Refraction
Oil minimizes refraction to capture more light with the objective lens

Your microscope
Let me demonstrate proper carrying of the microscope
Carrying your microscope to your bench
Your seat # = your microscope #
Let’s get our microscopes (located in cabinet next to you)

Microscope parts and their function
Ocular – provides magnification
Objective – provides magnification
Stage – holds the specimen; enables movement of the specimen
Condenser – concentrates or focuses light to illuminate the specimen
Iris diaphragm – changes the size of the cone of light, thus adjusting the amount of light
Coarse focus knob – raises & lowers the stage in larger increments, to adjust the focus
Use ONLY with scanning & low power objectives
Fine focus knob – raises & lowers the stage in smaller increments, to adjust the focus

Stage – holds the specimen; enables movement of the specimen
Microscope parts and their function
Stage
Stage clip

Tips for Today – Using Immersion Oil
Use oil only with the oil immersion objective (100X)
Get the specimen in focus under any lower power (4X or 10X) objective
Swing that objective out of the way, add a small drop of oil right on the specimen, and swing the oil immersion objective into place-oil will contact 100X objective
Don’t move the stage down to add oil
Use only the fine focus knob with oil immersion
Clean up all oil with lens paper
Objective, stage, specimen, etc.

Tips for Today – Drawings
Purpose of drawings is to cause you to carefully observe what you’re seeing
Draw a representative sample
This is not art class

Every drawing must be labeled with the name of the specimen and the total magnification
For example: yeast suspension, 100X

Tips for Today – Returning your microscope to the cabinet
Remove slide
Clean off all oil, using lens paper
Engage the lowest power objective lens (4X)
Wrap cord neatly around the base
Replace dust cover
Place scope carefully on the shelf, with oculars spun around and arm outward

Expectations: work as individuals
Examine a slide of letter “e”
Mount so “e” appears correctly as you see the slide on the stage

Examine prepared slide of colored threads
Examine using low power (10X) & high power (40X) objectives

Examine a prepared slide of bacteria
Examine using scanning, low power, & high power objectives
e

Why make smears and stain bacteria?
Looking at a bacterium under the microscope may be one of the first methods used to identify the cause of an infection
Direct exam of specimen
After culture of specimen

What’s the limitation of brightfield microscopy?
Bacterial cells are very small and lack contrast.
With brightfield microscopy, a stain is used to add contrast and make them visible.

Preparing Specimens for
Light Microscopy – The Smear
A smear is a thin film of microbes on a slide.
From broth: A loopful of broth culture
From slant/plate: A small amount of a colony is added to a very small drop of water
Let the slide air-dry
A smear is heat-fixed in order to:
attach the microbes to the slide
kill the microbes
increase stain penetration.

Types of Stains used by Microbiologists
Simple Stains – a single dye is used to add contrast, so microbes can be viewed microscopically
Positive stain
Negative stain
Differential Stains
Gram stain
Acid-fast stain
Special Structure Stains
Capsule stain
Spore stain
Flagellar stain

Simple Stains
Positive stain
Basic (positively-charged) dye interacts with negatively-charged bacterium.
Colored bacterium appears in a bright field.
Negative stain
Acidic (negatively-charged) dye is repelled by negatively-charged bacterium.
Stains background and leaves cells unstained.

Positive and Negative Staining
Positive stain
Negative stain

Making a Bacterial Smear (Ex. 3.5, pg. 186-187)

When observing a stained smear, you should always observe & record:
Shape
Arrangement
Size (a comment, if you can)
Don’t forget to label every drawing
What information in the label?
Specimen, total mag, staining procedure
Reminder: total magnification =
ocular (10X) X objective (4X or 10X or 40X or 100X)
Simple stains

coccus bacillus spirillum
Bacterial Shape

Singly
Pairs: diplococci, diplobacilli
Clusters: staphylococci
Chains: streptococci, streptobacilli
Arrangement

Stay organized with your label & observations
Tip for Today: Drawings
Name of bacterium
1000X
Simple stain with safranin
bacillus in
singles
singles
bacillus
1000X
Simple stain
Name of bacterium
safranin

For Next Lab:
Read: Ex 2-2 Colony Morphology and
Ex 1-3 Aseptic Transfers & Inoculation Methods (pgs. 29 – 48)

Ex 3-7: The Gram Stain
Pages 195-202

Today we will do 3 things:
Observe the 2 streak plates and assess the broth culture and slants from Tuesday for growth patterns
Gram stain some reference organisms
Observe our environmental plates on dissecting scope for Colony Morphology (Ex. 2-2)

Tools to characterize and identify microorganisms
Microscopic morphology
Colony morphology
Culture characteristics
Biochemical activities
Each of these is determined by an organism’s genes, and will be consistent for a bacterial species or isolate

Culture Characteristics in Broth
Surface:
Ring, pellicle, none
Sediment:
present, absent
Broth Clarity:
Uniform turbidity, flocculent *, clear
Amount of growth:
0 to +3 (none/slight/moderate/abundant)
*

Growth Patterns in Broth
pellicle*growth on surface
flocculent (clumps)
uniform turbidity
sediment
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Serratia marcescens. Gram negative, short bacilli that produce mucoid colonies which have entire margins and umbonate elevation. Note that there are both red and white colonies present on this plate. Some strains of S. marcescens produce the red pigment prodigiosin in response to incubation at 30o C, but do not do so at 37o C. This is an example of temperature-regulated phenotypic expression.
Broth: turbid appearance of the broth and the red color present in both the sediment and pellicle.
Slant: white to red color, smooth, moist growth

Micrococcus luteus. Circular, pinhead colonies which are convex with entire margins. This gram positive coccus produces a bright yellow, non-diffusable pigment.
Broth: produced granular and pellicle growth
Slant: abundant, opaque, grayish white, smooth, flat, slimy, and moist.

Escherichia coli. This gram negative, coccobacillus forms shiny, low convex, mucoid colonies which have entire margins and are slightly raised. Older colonies often have a darker center.
Broth: turbid growth with a deposit which disperse upon shaking.
Slant: off-white in color and showed a spreading growth pattern: usually it appeared as a cloudy film over the whole surface of the slant

Staphylococcus saprophyticus A Gram-positive, cocci forms slightly convex colonies that are opaque, smooth, circular, gray-white, white to cream colonies.
Broth: uniform turbidity with a fine to slight mucoid deposit.
Slant:

Culture Characteristics on a Slant
Slants are useful for keeping stock cultures of bacteria
Growth occurs on slant
The butt* allows for diffusion of nutrients to the organisms and diffusion of waste products away from organisms
Observe pattern of growth on the slant:

Note any other unique/obvious characteristics – e.g., color
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Purpose:
To determine appearance of growth on various media

This is consistent for an organism and is useful to characterize and identify organisms
Genetically determined, yet environmentally influenced
Compare organisms to see if they’re the same or different
Can tell us about their properties, such as the conditions/media at which they grow
3) Colony Morphology/Culture Characteristics

Colony Morphology/Culture Characteristics
7 Characteristics of Colony Morphology:
Shape
Margin
Elevation
Size
Color
Texture
Optical Properties

Shape

Elevation

Margin

Colony Characteristics (p. 64)

Size – measure in mm (or a comment)
Color – describe the color (specifically)
Texture – typically refers to the colony surface or its texture when probed with a loop
Optical Properties – how it transmits light (opaque, translucent, transparent)
Dissecting scopes are useful to observe colonies
Colony Characteristics

Summary: Characteristics to observe
On a plate
(as an isolated colony)
Colony Morphology
Shape
Margin
Elevation
Size
Color
Texture
Optical properties
On a slant
Growth on slants
Pattern of growth
Anything else that’s obvious or unique
In a broth
Growth in Broth
Surface
Sediment
Broth
Amount of growth

Streak Plate
Last time you created a streak plate from a mixed broth culture (Namely, E. coli, M. luteus, and Serratia marcescens)
A good streak plate:
Is appropriately labeled
Has an appropriate pattern
for streak lines
Uses the entire plate
Has well-isolated colonies
Is free of contamination

Did you obtain individual colonies?

Observe & critique your streak plate
To get more well-isolated colonies:
Use a small amount of initial bacterial inoculum (in first quadrant).
Remember to flame loop after each quadrant.
Don’t go back into previous quadrant so much (2 or 3 pass throughs).
Keep streak lines moving in forward direction.
Use entire plate.

Differential stains detect differences between organisms
The Gram stain differentiates bacteria into two groups: gram-positive and gram-negative, based on differences in cell wall structure.
Gram-positive bacteria tend to be killed more easily by antibiotics like penicillin.
Gram-negative bacteria can be more resistant to antibiotics.
Differential Stains: Gram Stain

Will react in a crystal violet dye and will stain dark purple or violet.
Cell wall is unique because it is composed of several peptidoglycan layers (multilayered) making it thicker.
Presence of techoic acids
Lacks an LPS (lipopolysachharide) content, periplasmic space and outer membrane making this group low in both lipoprotein and lipid composition.
Gram Positive

Normally does not react to a crystal
violet dye, stains pinkish red [decolorized in accepting safranin (a counterstain)]
Thinner peptidoglycan cell wall layer because it is just made of one single layer as opposed to gram positive’s multi-layered wall
Lacks teichoic acids, but has a high content of LPS
Has both a periplasmic space and an outer membrane
Has a high amount of lipoprotein content as well as lipids.
Gram Negative

Gram Stain
Start with an air-dried, heat-fixed smear.
primary stain mordant decolorizer counterstain

Crystal violet-iodine complexes form in cell
Gram-positive bacteria
Alcohol dehydrates & condenses thick peptidoglycan layer
Crystal violet-iodine complexes do not leave

Gram-negative bacteria
Alcohol dissolves outer membrane (LPS) and leaves holes in thin peptidoglycan layer
Crystal violet-iodine washes out
Gram Stain Mechanism

Differential Stains: Gram Stain
For every Gram stain, observe & record:
Gram reaction
cell shape
cell arrangement
size (if you can)

Gram Stain – Common Problems
Smears are too thick
Decolorizing too little or too much
Old cells and dead cells tend to stain Gram negative
Because the cell wall is not intact
Should always use fresh cultures

Demonstration of a Gram stain
Air-dried, heat-fixed smear
Remember: small amount of organism spread out in a small drop of water

Gram stain procedure – p 200
Decolorization is the most crucial step

Until run-off is clear
page 200

Expectations: Each student should:
Observe & critique your streak plates
Prepare & Gram stain 1 slide
Slide: Three smears on slide:
Gram Pos., Mixed, and Gram Neg.

Gram stain procedure – p 200

Observe with oil immersion objective (100 X)
Observe simple stained slides from last lab and colony morphology

S. Sapro
Or M. luteus
S. marcescens
Or E. coli
Mixed

coccus bacillus spirillum
Bacterial Shape

Singly
Pairs: diplococci, diplobacilli
Clusters: staphylococci
Chains: streptococci, streptobacilli
Arrangement

For Next Lab:
Read:
Ex 3.9 Capsule Stain
Ex 3.10 Endospore Stain
Ex 3.12 Flagella Stain

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