Overview Overview

Turn in Hamburger ReportsTurn in Hamburger ReportsQuizQuiz Discuss Bacterial Growth ReportsDiscuss Bacterial Growth Reports Pre-lecture for Exercise 7: Phage One-Step Pre-lecture for Exercise 7: Phage One-Step Growth Growth

ExperimentExperiment Work on Environmental Isolate-Facultative Work on Environmental Isolate-Facultative anaerobes (Section E tonight, Section D Friday)anaerobes (Section E tonight, Section D Friday)

Bacterial Growth ReportBacterial Growth ReportIntroductionIntroduction

Paragraph 1Paragraph 1 Description of bacterial growth (phases)Description of bacterial growth (phases) Background informationBackground information

Paragraph 2Paragraph 2 Objective: why did we do this experiment?Objective: why did we do this experiment? General procedure: how did we accomplish the General procedure: how did we accomplish the

objective?objective?

Paragraph 3Paragraph 3 Hypothesis, predictionsHypothesis, predictions Supportive rationaleSupportive rationale

Materials and MethodsMaterials and Methods

Past tense, Passive voice:Past tense, Passive voice: The boy caught the dog. (correct tense, wrong The boy caught the dog. (correct tense, wrong

voice)voice) The dog was caught by the boy. (correct)The dog was caught by the boy. (correct)

Do not simply list the methods: Do not simply list the methods: put in your own words in paragraph formput in your own words in paragraph form

Chronological orderChronological order

ResultsResults

Table of ODTable of OD550550 and time and time

Excel Graph of dataExcel Graph of data

0.01

0.1

1

0 20 40 60 80

Time (min)

OD

550

20% Glucose

YE-P

Expon. (YE-P)

Expon. (20%Glucose)

Results cntd.Results cntd.

Hand drawn graph:Hand drawn graph: Axis labeled Axis labeled All data pointsAll data points Best fit linesBest fit lines Lines indicating how you got the generation Lines indicating how you got the generation

timetime

Results cntd.Results cntd.

What you should have seen:What you should have seen: Doubling time should decrease by about halfDoubling time should decrease by about half Ex. 60 min glucose, 30 min YEPEx. 60 min glucose, 30 min YEP

What to do if your results were different:What to do if your results were different: Talk about why your results are different in Talk about why your results are different in

the discussion.the discussion.

DiscussionDiscussion

Paragraph 1Paragraph 1 Summarize the report objectives and Summarize the report objectives and

hypothesishypothesis Were the objectives accomplished?Were the objectives accomplished?

Paragraph 2Paragraph 2 Analyze data: what does the data mean, why Analyze data: what does the data mean, why

is it important?is it important?

DiscussionDiscussion

Paragraph 3Paragraph 3 Does your data support your hypothesis?Does your data support your hypothesis? Are there possible errors? Are there possible errors?

Think outside the box…errors other than human Think outside the box…errors other than human errors.errors.

Paragraph 4Paragraph 4 Compare results to other relevant studiesCompare results to other relevant studies

Literature CitedLiterature Cited

4 total sources4 total sources 3 journal articles 3 journal articles Lab manualLab manual You can have more sources, but not lessYou can have more sources, but not less

Use citation handout for formattingUse citation handout for formatting

What is the mistake on this slide?What is the mistake on this slide?

Exercise 7Exercise 7

We’ll be working with a bacteriophage next We’ll be working with a bacteriophage next week in classweek in class

This is another that takes the entire timeThis is another that takes the entire time

Pre-lecture this weekPre-lecture this week

Next week a quick overview of the Next week a quick overview of the procedure as a reminderprocedure as a reminder

So what is a bacteriophage?So what is a bacteriophage?

A virus that infects bacteriaA virus that infects bacteriaSize: T4 phage is 85x110 Size: T4 phage is 85x110 m (m (E. coliE. coli cell cell is about 1x3 is about 1x3 m) m) Brock Biology of Microorganisms, 11Brock Biology of Microorganisms, 11

thth ed. ed.

Importance:Importance: General viral understandingGeneral viral understanding Killing cells you want them toKilling cells you want them to Killing cells you don’t want them toKilling cells you don’t want them to

Bacteriophages are often considered the Bacteriophages are often considered the most numerous entities on the planetmost numerous entities on the planet

Structure of BacteriophageStructure of Bacteriophage

Head or CapsidHead or Capsid IcosahedralIcosahedral Protective covering for the nucleic acidProtective covering for the nucleic acidTailTail A hollow tube through which the nucleic acid A hollow tube through which the nucleic acid

passes during infection.passes during infection. Surrounded by a contractile sheath.Surrounded by a contractile sheath. Base PlatesBase Plates Tail fibersTail fibers

A package of A package of DNA/RNADNA/RNA

A transport tubeA transport tube

An attachment An attachment

devicedevice

Stages of Host Cell InfectionStages of Host Cell Infection

AdsorptionAdsorption Attach to cell, inject DNAAttach to cell, inject DNA

ReplicationReplication Phage DNA takes over host cellPhage DNA takes over host cell

AssemblyAssembly Mature phage particlesMature phage particles

LysisLysis Host cell bursts and releases mature Host cell bursts and releases mature

phagephage

Adsorption

Assembly

Lysis

Replication

Phage InfectionPhage Infection

AdsorptionAdsorption AttachmentAttachment

Maturation periodMaturation period Assembly of phage particlesAssembly of phage particles

Eclipse period or Latent PeriodEclipse period or Latent Period No mature phage particlesNo mature phage particles Time between initial infection and release Time between initial infection and release

of mature phageof mature phage

Phage Growth CurvePhage Growth Curve

Phage Growth CurvePhage Growth Curve

Rise periodRise period Host cells begin to burstHost cells begin to burst

PlateauPlateau Constant number of phage near the end Constant number of phage near the end

of reproduction cycleof reproduction cycle

Burst sizeBurst size Average number of phage particles Average number of phage particles

released by each infected bacteriumreleased by each infected bacterium

http://www-micro.msb.le.ac.uk/224/Phages.html

T4 Phage AssayT4 Phage Assay

Set-UpSet-Up Use Figure 7.1 to arrange and label 15 Use Figure 7.1 to arrange and label 15

culture tubesculture tubes Label w/ time and rowLabel w/ time and row Label dilution tube and place in water bathLabel dilution tube and place in water bath Transfer appropriate amount of LB broth to Transfer appropriate amount of LB broth to

each tubeeach tube Label plates (min., initials, date, experiment)Label plates (min., initials, date, experiment)

Phage One-Step Growth Phage One-Step Growth ExperimentExperiment

© Phage et al.

Phage One-Step Growth Phage One-Step Growth ExperimentExperiment

9.9 9.9 9.9 9.9 9.9 9.9 9.9

9.9 9.9 9.9 9.9 9.0 9.0 9.0

Row 1

Row 2

15 25 30 35 40 45Time(min)

50

Phage One-Step Growth Phage One-Step Growth ExperimentExperiment

AdsorptionAdsorption Place phage in 37°C Place phage in 37°C

water bath for 1-2 water bath for 1-2 minutesminutes

Vortex Vortex E. coliE. coli culture culture Transfer 2.9 ml if Transfer 2.9 ml if E. coliE. coli

into ADS tube w/ phageinto ADS tube w/ phage THIS IS TIME THIS IS TIME

ZERO!!!!!ZERO!!!!! Finger vortex ADS tube Finger vortex ADS tube Fill in time table in lab Fill in time table in lab

bookbook

Phage One-Step Growth Phage One-Step Growth ExperimentExperiment

Dilution to stop adsorptionDilution to stop adsorption After five minutes gently mix ADS tubeAfter five minutes gently mix ADS tube NEVER VORTEX ANY TUBE CONTAINING NEVER VORTEX ANY TUBE CONTAINING

PHAGE!!!!!!!PHAGE!!!!!!! Transfer 0.1 ml from ADS tube to Dilution tubeTransfer 0.1 ml from ADS tube to Dilution tube Return to 37°C water bath until next time pointReturn to 37°C water bath until next time point

(This step dilutes the phage and E. coli to the point that (This step dilutes the phage and E. coli to the point that there are very few collisions and adsorption does there are very few collisions and adsorption does not occur)not occur)

What will happen if you vortex What will happen if you vortex phage (other than finger vortex):phage (other than finger vortex):

Phage will be spliced into little bits!!!

Phage One-Step Growth Phage One-Step Growth ExperimentExperiment

Taking 15, 25, and 30 Taking 15, 25, and 30 min. samplesmin. samples

After five minutes gently mix After five minutes gently mix dilution tubedilution tube

Transfer 0.1 ml from Dilution Transfer 0.1 ml from Dilution tube into the 1tube into the 1stst tube in your tube in your rackrack

Gently mixGently mix Transfer 1.0 ml from 1Transfer 1.0 ml from 1stst tube to tube to

the tube behind itthe tube behind it Gently mixGently mix

Phage One-Step Growth Phage One-Step Growth ExperimentExperiment

Get a tube of soft agar from 47°C water bath Get a tube of soft agar from 47°C water bath Add 0.3 ml of Add 0.3 ml of E. coliE. coli B, lawn cells to soft B, lawn cells to soft

agar and vortex BRIEFLYagar and vortex BRIEFLY Transfer 0.1 ml from 2Transfer 0.1 ml from 2ndnd row tube to soft row tube to soft

agar agar Pour Pour IMMEDIATELYIMMEDIATELY on to plate labeled on to plate labeled

‘‘15 min’ and use figure 8 to spread15 min’ and use figure 8 to spread

Phage One-Step Growth Phage One-Step Growth ExperimentExperiment

Taking 15, 25, and 30 min. SamplesTaking 15, 25, and 30 min. Samples After five minutes gently mix Dilution tubeAfter five minutes gently mix Dilution tube Transfer 0.1 ml from Dilution tube into the Transfer 0.1 ml from Dilution tube into the

1st tube in your rack1st tube in your rack Gently mixGently mix Transfer 0.1 ml from 1st tube to the tube Transfer 0.1 ml from 1st tube to the tube

behind itbehind it Gently mixGently mix

Phage One-Step Growth Phage One-Step Growth ExperimentExperiment

Get a tube of soft agar Get a tube of soft agar from 47°C water bath from 47°C water bath

Add 0.3 ml of Add 0.3 ml of E. coliE. coli B B lawn cells to soft agar and lawn cells to soft agar and vortex BRIEFLYvortex BRIEFLY

Transfer 0.1 ml from 2nd Transfer 0.1 ml from 2nd row tube to soft agar row tube to soft agar

Pour Pour IMMEDIATELYIMMEDIATELY on to on to plate labeled plate labeled

‘‘15 min’ and use figure 8 15 min’ and use figure 8 to spreadto spread

Once soft agar has solidified, tape plates Once soft agar has solidified, tape plates together and incubate at 37°C for 24-48 together and incubate at 37°C for 24-48 hourshours

During lab next week we will count the During lab next week we will count the plaques and fill out the worksheetplaques and fill out the worksheet

Worksheet will be due at the end of lab Worksheet will be due at the end of lab next weeknext week

Phage One-Step Growth Phage One-Step Growth ExperimentExperiment

Plaques = clear areas on a lawn of host cells that represent the point at which a single infectious

virus particle was deposited As a result of subsequent lytic cycles, the host cells in

the region are destroyed.

Phage One-Step GrowthPhage One-Step Growth

Phage One-Step Growth Phage One-Step Growth ExperimentExperiment

Plaque formation is analogous to the plate Plaque formation is analogous to the plate count of bacteria in which each plaque count of bacteria in which each plaque (colony) represents a single infectious (colony) represents a single infectious virus (viable bacterium) in the initial virus (viable bacterium) in the initial inoculum. inoculum. A bacteriophage plaque count can be A bacteriophage plaque count can be used to determine the used to determine the titertiter, the , the concentration of phage particles, in the concentration of phage particles, in the suspension provided.suspension provided.

The Experiment The Experiment The Step by StepThe Step by Step

ADS

TIME ZERO2.9 ml E. coli Binto phage T4

Phage T4

Water Bath37ºC

9.9 9.9 9.9 9.9 9.9 9.9 9.9

9.99.99.99.99.09.09.0

Row 1

Row 2

15 25 30 35 40 45Time(min)

50

5 min later0.1 ml

DIL

9.9

1:100

20

9.9

1:10,000

9.0

1:100,000

0.3 ml E. coli B

lawn cells

4 ml Soft Agar47ºC

15

25

45

40

9.9

1:10,000

9.0

1:100,000

35

50

0.1 ml

0.1 ml

0.3 ml E. coli Blawn cells

4 ml Soft Agar47ºC

0.1 ml

1.0ml

1:1,000,000

1:10,000,000

0.1 ml

Environmental Environmental IsolateIsolate

If time permits, continue working on If time permits, continue working on identifying your environmental isolate:identifying your environmental isolate:

- - Determine storage Determine storage conditionsconditions (you must maintain your (you must maintain your organism for the rest of the semester)organism for the rest of the semester)

- - Biochemical testsBiochemical tests- - Bergey’s ManualBergey’s Manual- Additional tests- Additional tests

Next WeekNext Week

Ex. 7 is another one that will take the entire time Ex. 7 is another one that will take the entire time so be here, ready to start on time!so be here, ready to start on time!

First submission of bacterial growth report dueFirst submission of bacterial growth report due Remember-ALL sections have to be attemptedRemember-ALL sections have to be attempted Don’t treat this as a rough draftDon’t treat this as a rough draft

We will fill out the phage worksheet in class and We will fill out the phage worksheet in class and you will hand it inyou will hand it in You may want to look it over this week and start filling You may want to look it over this week and start filling

in some definitionsin some definitions