Make LPGM

with 5%, 7%, and 10% sucrose

Incubate briefly in LPGM in a 2 mL tube in the Ferris wheel, at a slow spin speed, parallel to the direction of rotation so the tube goes upside down and the liquid moves from top to bottom of the tube.

Can image at 4x on a poly-lysine coated slide . Make a vaseline circle, put some pollen suspension inside it, cover with coverslip.

Set up time-lapse imaging: 20 images every 5 minutes. Find a field of view with several pollen grains in focus at once, all starting to germinate.

In a multiwell plate The surface of one well of a 12 well plate can be covered by as little as 400uL water.

Need to cover with poly-lysine. Already have ~50 mL of 0.1% working solution.

So can coat ~125 wells

Well diameter = ~22 mm

In a cover-slip bottom 60 mm dish

500 uL coats the well.

Coverslip diameter = 30 mm

How many unique fields of view can we get in one dish?

Pollen tubes grow ~0.2 um/sec = 12um/min = 0.012 mm/min

Reasonably good optics at 10x; camera adds magnification.

Coat all wells on 3 12-well plates, one for each concentration of sucrose.

Bacterial plasmids:

• H2B-mCherry (Addgene 20972) (Kan resistant)
• ecadherinGFP (Addgene 28009) (Kan resistant)

Restriction Enzymes

• PvuI-HF (cut smart)
• BamHI-HF (cut smart)
• KpnI-HF (cut smart)

A "Typical" Restriction Digest

Gel Loading

• 1 % agarose in TAE
• 1/10,000 SYBR Safe
• 20 wells
• wide gel
• 10 uL/well

Lanes 1, 10, 20 have NEB 1Kb ladder mixed in this ratio

• 1 uL ladder
• 1 uL loading dye
• 6 uL water

Restriction sites

Digest fragments

Molecular biology PowerPoint from 2020 remote class

Grow bacteria

Streak frozen cells on agar plate

Pick a colony and grow overnight in liquid LB (plus antibiotic if necessary)

Concentrate ON culture in centrifuge

Make 2 dilution series: 10-fold, 2-fold

• 2 fold

  • tubes 1A - 10A
  • 0.5 mL LB in each
  • add .5 mL culture to first, mix thoroughly
  • serially dilute by moving 0.5 mL along the series*

• 10 fold

  • tubes 1B - 10B
  • 0.9 mL LB in each
  • add .1 mL culture to first, mix thoroughly
  • serially dilute by moving 0.1 mL along the series

Take OD measurements

• Load 250 uL of each dilution into 96 well plate
• samples in 1st 10 wells of 1st 2 rows
• LB in last 2 wells
• Read, using bug OD

Plot results

• Put fraction of original culture next to each OD
• Cut, paste special, values only
• Sort data
• Make a scatterplot
• Find the lowest concentration just above the noise for hemocytometer

Hemocytometer

• Load 10 uL into hemocytometer.
• Photograph.
• Also take photographs of too concentrated and too dilute samples

Colony Counts

• spread 100 uL from last 5 10-fold dilutions on agar plates
• Grow overnight
• Photograph

Part 1 of PowerPoint from 2020 remote lab

Part 2 of PowerPoint from 2020 remote lab

Qualitative lac operon experiment

Grow GFP E. coli in different sugar media, then photograph in a 12 well plate on a blue light box through the orange filter.

• Streak GFP E. coli on an LB-glucose-kan plate, grow overnight

• Pick a colony, grow in overnight in liquid LB-glucose-kan (to prevent it turning green)

• Spin down the ON culture (take a picture of the pellet for other use!)

• Rinse several times (to get rid of the glucose)

• Fill 12 well plate as indicated below. 2 mL per well:

• Photograph
  • in white light
  • in blue light

• Read in plate reader for blanks (OD and FL)

  • 12-well plate is too tall for reader!!!
  • Sample 100 uL of each to read blanks (OD & FL in a 96 well plate)

• Inoculate each well with a small amount of culture.

• Incubate on the shaker.

• Photograph

  • in white light
  • in blue light
• Read in plate reader (OD and FL)
  • Take 100 uL (?) samples to read in 96 well plate

Media

Need enough for the qualitative experiment ~5 mL each

Need enough for the quantitative experiment: 96 well plate @ 250 uL/well = ~25 mL total

Make 25 mL of each high concentration stock; mix the combinations from that

Add 50 µg/mL Kanamycin as indicated here.

Lac Operon PowerPoint from 2020 remote class

For 9/2 & 9/7 2021
For 9/6 & 9/8 2022

Lab 1.1 protocol

• Pipetters & tips
• trash bucket
• Balances
• water beaker
• liquid waste bucket
• gloves
• Fluorescein 1 mM (~5 mL)
• 12 glass tubes in rack
• white and black strips behind rack
• yellow filters
• blue flashlights

9/9 & 9/14 2021

Lab 1.2 protocol

• Getting files from Wahoo.

• Link to bcrc accounts

• tube to mix 2 mL

• rack to hold tubes: fluorescein stock tube, NaOH tube, dilution tube

• 1 mM fluorescein in 10mM NaOH

• 10mM NaOH

• 96 well fluorescence plate

• tape & marker

• liquid waste beaker

• tips & other dry trash beaker

9/16 & 9/21 2021

• tube to mix 2 mL

• rack to hold tubes: fluorescein stock tube, NaOH tube, dilution tube

• 1 mM fluorescein in 10mM NaOH

• 10mM NaOH

• 96 well fluorescence plate (can use saved plate)

• tape & marker

• liquid waste beaker

• tips & other dry trash beaker

• two unknown fluorescein concentrations (one too dilute for Abs, one too concentrated for FL)

9/16 & 9/21 2021 9/20 & 22 2022

2020 experiment

Bacterial plasmids:

• H2B-mCherry (Addgene 20972) (Amp resistant) (Labeled A)
• ecadherinGFP (Addgene 28009) (Amp resistant) (Labeled B) GROWS SLOWLY! ALLOW EXTRA TIME!

Miniprep 2022

• Tuesday: Zyppy (use up old aliquots)

• Thursday: ZR Classic

Mini-Prep 2021

• micro pipetters & tips
• labeled racks in fridge
• 1 mL each E. coli culture (concentrated by centrifugation) per pair
• centrifuges

• Miniprep kits - aliquot miniprep reagents per pair:

  • sterile microfuge tubes
  • 250 uL Buffer P1(+ RNaseA+LyseBlue)/rxn = 510 uL per pair
  • 250 uL Buffer P2/rxn = 510 uL per pair
  • 350 uL Buffer N3/rxn = 710 uL per pair
  • 2 QIAprep spin columns
  • 0.5 mL Buffer PB/rxn = 1.1 mL/pair
  • 0.75 mL Buffer PE/rxn = 1.51 mL
  • 50 uL Buffer EB (10 mM Tris-Cl pH 8.5) per rxn = 110 uL per pair
  • sterile water

Restriction Digest

9/27 & 9/29 2022 (9/23 & 9/28 2021)

2020 experiment

Reagents & Materials

• 2 plasmids from Lab 2.1

• Wide format gel

  • 6 1% 100 mL SYBR Safe gels
  • TAE in carboy
  • 2 20-well combs per gel (buy more!)
• Restriction enzymes (get the large size!)
  • BamH1-HF
  • Kpn1-HF
  • Pvu1-HF
• Diluent B to dilute enzymes to 1/4 strength
• NEB Purple 1 Kb ladder
• Purple loading dye SDS-free (comes with enzymes)
• hot blocks for 168 reactions (!) Maybe an incubator?

Restriction Digest Volumes

Aliquots

16 reactions per pair (8 reactions x 2 plasmids):

• 3 single
• 3 double
• 1 triple digest
• 1 uncut

• For 3 single, 3 double, 1 triple digest, and 1 uncut x 2 plasmids per pair:
  • Cut smart: 50 µL (16 rxns x 2.5 µL = 40 µL)
  • Enzymes: dilute to 1/4, then give 20? µL (4 rxns x 4 µL = 16 µL)

Restriction sites

Digest fragments

9/30 & 10/5 2021

10/7 & 10/12 2021

Wet materials:

• LB plates, 8 per pair = 96/section
• LB broth (cold) ~10 mL/pair
• E. coli at stasis: 3 mL aliquots in round bottom tubes, in fridge, 1 per pair = 36 mL
• ice in small container

Equipment

• hemocytometers
• glass beads
• beaker for used beads at each table
• tips beaker
• liquid waste beaker
• microfuge tubes & rack
• marker
• clear 96 well plate
• pipetters & tips

Plate reader program

• BUG_OD
• 1st 2 rows

10/14 & 10/19 2021

Give them the Excel growth curves.

Consider doing a 37C growth curve as well.

10/25 & 10/27 2022: Combined with 4.2

Make 1 set of 12 cultures for each table.

10/21 & 10/26 2021

• sterilize 12 small flasks for cultures
• sterilize at least 288 glass culture tubes

See prep for 2015

See LB sugar recipes

• Make 50 mL of each mixture
• Put 25 mL into flask for shaking incubator
• Put 1 mL into microtube for blanks.
• Save the rest for the second section (in case you need to grow more cells).
• Make 12 (or 6) 2 mL aliquots - one set per group; 5 mL aliquots of LB

See E. coli for Lac operon

Spread glucose + Kan on plain LB plate, then

Streak incubate overnight, and pick a colony into each medium.

Allow to grow, but not too long! Over day, but not over night? Overnight is OK, but with temp below 30C

Put out blue flashlights and yellow filters

Class can share one black fluorescence plate

10/28 & 11/2 2021

Make 12 tubes of 12 media, 5 mL each.

6 sets per day.

Day before:

• make LB-glu (mix 20% glucose from genetics 1:1 with LB if necessary)
• Start overnight culture in LB-glu

Day of:

• Borrow Biochem multi-channel pipet plus small tips. Dial down to 5 uL
• Subculture into LB glu again
• Just before use, wash with LB 3x
• Resuspend in 10 mL LB
• Need OD = 0.02 in the wells
  • Need 10 mL to put in sterile petri dish to fill pipet
  • Need an OD that doesn't appreciably raise the OD of 205 uL LB (OD =~0.2)
  • Aim for blank corrected OD of 0.8
  • Dilute as necessary

11/4 & 11/9 2021

11/18 & 11/30 2021

11/18 Thursday

Group 1: Botros et al.

• Zone of inhibition
• E. coli Addgene 20972 from molecular biology experiment
• LB Amp 25 mL
• alcohol (ethanol or isopropanol?)
• disinfectant??
• 20 LB amp plates for zone of inhibition
• sterile discs
• sterile forceps

Group 2 (Vengalil et al.):

• E. coli Addgene 20972 from molecular biology experiment grown in LB-amp
• LB amp salt-free
• LB amp 2M NaCl
  • add 2.922 g NaCl per 25 mL salt-free LB
• LB amp 2M KCl
  • add 14.9 g KCl per 100 mL LB
• 20 LB amp plates

Group 3 (Long, et al.):

• 40 LB plates (no antibiotic)
• Replica plate materials
• Kid's hand sanitizer
• room swipe access (need names and Spire IDs)

Group 4 ( Lee, et al.):

• E. coli GFP culture in LB Kan
• LB Kan ~25 mL
• LB-glu Kan ~15 mL
• 96 well black plate, shared with group 5
• rewrite script for only the rows they use

Group 5 (Creto, et al.)

• E coli GFP culture in LB Kan
• LB Kan ~25 mL
• LB-glu Kan ~15 mL
• LB-lac Kan ~15 mL
• LB-gal Kan ~15 mL
• 96 well black plate, shared with group 4
• rewrite script for only the rows they use

11/30 TUESDAY

McKnight, Carson, Matfes, Berke

• change pH of LB
• GFP expression
• plate reader

Bowen, Howall, Abraham, Wald

• fertilizer - N-based vs P-based.
• Nitrogen vs phosphorus fertilizer
• LB,
• sterile tubes,
• plates
• Culture (non-GFP?)

Boyd, Ebian, Melusen, Manning

• pH again

Cloutier, Dawiczyk, Tan, Joyce

• mouthwash
• plate reader
• culture (non-GFP?)
• Liquid LB

Alexander, Ferriera, Beinstein, Birnbach

• comparison of various substances at killing E. coli
• LB broth and plates
• E coli culture (not GFP?)

Crotty, Galligan, Horrigan, Jansen

• NaCl
• KCl
• plate reader overnight
• agar plates

11/23 (Thursday section) & 11/30 2021

12/2 & 12/7 2021

Materials

• one balance per table (borrow from Bill Schmitt)
• weighboats
• non-sterile tips
• Mats
• Gloves
• small glass culture tubes (~15/pair)
• rack for tubes

• beaker for water

• blue flashlights & yellow filters

Computers and Plate Readers

• Check program Repeatability (Abs 485 nm; layout A1-B12)
• Wahoo folders must be named Tues01, Tues02, ...Tues12 and Thur01, Thur02 ... Thur12, even though the user names in the Optima program are Tue01 and Thu01, etc.
• Import program to groups
• Check network connections (in “my computer” connect the Z drive to Wahoo as kdorfman, kd@ISB)

10mM NaOH

MW = ~40

10mM = 0.01M = 0.4g/L

1 mM Fluorescein

in 10 mM NaOH

It fades with time, so check the concentration of the old stocks.

MW = 332

1mM = 0.332g/L = 0.033g/100 mL

A = Ecl

Molar extinction coefficient of fluorescein at 485nm = 50358/mol.cm

so Absorbance 10^-5M = 0.50358

(1/100 of a 1mM solution =10^-5M)

Absorbance of Fluorescein

Per pair:

• Black 96 well plate (not necessarily sterile, but clean)
• 1 mM fluorescein (~ 1 mL)
• 10 mM NaOH (~ 4.5 mL)
• 2 2-mL tubes to make 2 mL of 10µM fluorescein (each partner does one)

Computers and Plate Readers

• Check program Repeatability (Abs 485 nm; layout A1-B12, volume 150 µL)
• Check program FL-Abs-by-2 (Abs 485 nm; layout C1-2, D1-12, volume 150 µL)
• Wahoo folders must be named Tues01, Tues02, ...Tues12 and Thur01, Thur02 ... Thur12, even though the user names in the Optima program are Tue01 and Thu01, etc.
• Import program to groups
• Check network connections (in “my computer” connect the Z drive to Wahoo as kdorfman, kd@hh3)

Corrections

Read the plate only once

Pipet 150 µL of each unknown in E3 - 12 as needed

Programs

• Serial2&5_Abs
• Serial2&5_FL
• E1 &E2: blank E3-E12 for unknowns
• F1 - F12: dilute by 2
• G1 - G12: dilute by 5

Materials:

• ~1 mL 1mM fluorescein (try to make it right via Abs)
  • E = 50358/M
  • A = 50358/M * C
  • at 10 µM, A = 0.5038
  • dilute 1 mM 1/100 to make 10 µM, take Abs
• 5 mL 10 mM NaOH
• black plate from last time (or new one if old one has no clean wells)

Conversion factors

Abs = 59.7*(mM)-0.341 up to mM=0.03125

FL = 10^6 * mM +173.06 up to mM = 0.008

FL = 2 X 10^6 * mM + 19.624 up to mM = 0.00195

Unknowns

• Make dilution series from the "1mM" stock.
• find lowest useful concentration for Abs. Dilute slightly for unknown 1 - must be done with FL. Abs is bad below 0.0005 and meaningless below 5 x 10^-7mM
• find a concentration too high for fluorescence, but OK for abs, e.g., 0.05.
• find a concentration too high for either. must be >0.125

2013:

Start with ice cold culture in stationary phase, OD = ~1.5

Make a 2-fold dilution series (in microtubes?)

Make a 10-fold dilution series (in microtubes?)

Load 96 well plate

Get OD’s

Keep cells on ice!

Make OD vs dilution curve (combine data from 10-fold and 2-fold dilution series)

Count cells from some well (should have theoretical OD = ~ 10-6).

Too many? Use a more dilute one. Too few? Go up one.

Make agar plate from 8 wells

Calculate OD (from Od vs dilution factor) for the wells used in hemocytometer

Calculate OD for the wells used in the agar plate

Get cells/mL/OD conversion factors by both methods. Compare.

OR: make a table: if OD of first well is __, then use well# __ for the hemocytometer.

Important observations

A stationary phase culture (left at RT for several days) has OD = 2.6

Relationship between dilution and OD is linear through about OD = 1.5

By hemocytometer counts, there are ~5 x 10^8 cells/mL/OD

Cells are countable (19 cells/0.004 µL and 27 cells/0.005 µL) in the hemocytometer at 5 x 10^6 cells/mL, or blank corrected OD = ~0.01 (about where it slips into the noise)

So recommend either

• to start with well #9 in the 2-fold dilution series, or
• if they have done the OD's first, use the first well with a blank-corrected OD below 0.03

Week before lab:

• streak out control cells on agar plate

• grow overnight at 37C

• cover plate and refrigerate

Two days before lab:

• pick colony, put into enough LB for all students (~250 mL)

• grow on shaker overnight

Day before lab:

• put LB in fridge

  • need ~15 mL
  • 2013 - have lots of 25 mL tubes of LB. Use them up

• check during day for stasis (OD = ~2.6 in previous years; growth seems to stop at OD = ~2, which is still in the linear range)

NOTE: If left too long on shaker, get too many in chains. Check culture first thing in the morning with microscope. OD = 2, still swimming. Put in fridge.

• refrigerate to stop growth

• Dilute or spin down and resuspend to OD = ~2.5 (around max linear correlation to conc) with ice cold LB

• Aliquot ~4 mL/tube

• 1 tube per pair

Reagents

TUESDAY

Group 1

Replica plates, hand before and after treatment with:

• 48 LB plates
• antibacterial soap
• regular soap
• bleach (0.005%)
• hand sanitizer
• velvets
• stamp disks

Group 2

Zone of inhibition

• 10 LB plates
• ON culture (RP 437)
• filter disks
• sterile forceps
• miscellaneous poisons

Group 3

E. coli growth rate in min medium + sugars (overnight in plate reader)

• M9
  • as is (glycerol)
  • +0.4% glucose
  • • 0.4% galactose
  • • 0.4 % maltose
  • • 0.4 % lactose
• growing E. coli RP437 (chemotaxis wild type)

See here for source of 0.4% sugars

Group 4

E. coli growth rate in liquid medium plus drugs in plate reader

• E. coli culture (use the RP437 from the other group)
• 2x LB
• caffeine
• EDTA
• Ethanol

Group 5 E. coli growth rate at different pH

• growing culture
• 2x LB
• PBS? Tris? buffered at different pH
• Make LB in 10 mM Tris at pH 5.5, 6, 6.5, 7, 7.5, 8, 8.5, in glass tubes
  • Mix 1 M tris + Tris base to each pH,
  • 100 uL + 9.9 mL water in glass tube or vial
  • 0.25 g LB
  • autoclave

Group 6 Incubate E. coli in different concentrations of ethanol, then plate for colony counts

• 2x LB
• E. coli in liquid culture (RP 347)
• ethanol
• 50 plates

THURSDAY

Group 1

• 4 types of mouthwash (60 mL each) student supplied

• sterile swabs

• 24 LB plates

Group 2

Frat floors

• swabs

• 24 plates

• 4 types of disinfectants student supplied

Group 3

• cells

• 6 plates

• sterile disks

• sterile beads

• disinfectants (student or Laura supplied)

• sterile forceps

Group 4

Plate reader assay

• GFP-lac cells

• LB antibiotic liquid culture

• LB-lac

• LB- glu

• LB-lac-glu

• LB-mal

• LB-lac=mal

• LB

Group 5

OD plate reader experiment. (same as Tuesday Group 4)

• 2xLB
• caffeine
• alcohol
• EDTA

Group 6

Plat reader: Lac-GFP cells

• LB

• LB-sucrose

• LB-lac

• LB-glu

First thing, the morning before:

• Pick a colony
• Grow in 3 mL LB-Kan

Last thing, the day before: 100 µL culture into 40 mL (each w/ Kan)

• LB
• Lac 0.25 M
  • 20 mL LB
  • 20 mL Lac 0.5 M
• IPTG 0.1mM
  • 20 mL LB
  • 20 mL 0.2 mM IPTG
• Glu 0.25 M
  • 20 mL LB
  • 20 mL 0.5 M glu
• Gal 0.25 M
  • 20 mL LB
  • 20 mL 0.5 mL gal
• Mal 0.25 M
  • 20 mL LB
  • 20 mL 0.5 M mal
• IPTG 0.1 mM + glu 0.25 M
  • 20 mL 0.2 mM IPTG
  • 20 mL 0.5 M glu
• IPTG 0.1 mM + mal 0.125 M
  • 20 mL 0.2 mM IPTG
  • 10 mL LB
  • 10 mL 0.5 M mal
• Lac 0.25 M + glu 0.25 M
  • 20 mL 0.5M lac
  • 20 mL 0.5 M glu
• Lac 0.25 M + mal 0.125 M
  • 20 mL 0.5M lac
  • 10 mL LB
  • 10 mL 0.5 M mal
• Gal 0.25 M + glu 0.25 M
  • 20 mL 0.5 M gal
  • 20 mL 0.5 M glu
• Gal 0.25 M + mal 0.125 M
  • 20 mL 0.5 M gal
  • 10 mL LB
  • 10 mL 0.5 M mal

Morning of:

• Aliquot ~4 mL each culture into plastic tubes.
• One set per 4 tables.
• ? save for next lab in fridge?

Mixtures needed to grow 50 mL cultures for qualitative experiment

Need an additional ~120 mL of each sugar for quantitative student experiments the next week. (24 5-mL aliquots each)

LB-sugar recipes

Plus at least 100 mL for student experiments

To make 25 mL for each culture (6 sets of 12 4-mL cultures):

Make enough media for 3.1 (25 mL) and 3.2 (200 mL of LB, 150 Lac, 150 glu, 100 each of the others)

Calculate amount of each medium assuming they will make mixtures in 2 mL tubes. Each group needs ~12 mL. Mostly LB, then lac, then glu

for 3.1:

• Each pair gets 1 set of 12 tubes, each with E. coli already grown up. LB +

  • nothing
  • lac (0.25 M)
  • IPTG (0.01mM)
  • glu (0.25 M)
  • gal (0.25 M)
  • mal (0.25 M)
  • IPTG + glu
  • IPTG + mal
  • lac + glu
  • lac + mal
  • gal + glu
  • gal + mal

• Overgrown cultures all are too fluorescent!

• Can use the same tubes for both lab sections (if students don't throw them out)
• Night before: grow up GFP E coli from liquid culture in ~25 mL
• morning of: aliquot cultures into tubes 1mL each.

Which chemical compounds influence lac operon expression?

GFP E. coli grown in LB

Grow cells in LB-Kan ¹

Overnight culture

Dilute and monitor in morning; keep cells in active growing phase. Turn off heat in shaker if necessary.

Need ~20 mL total (8x12x2x100uL)

OD of culture should be 0.4 when cells are added to plates. That will put the OD just down into the noise, so there will be a lag time at the beginning of the run.

TA aliquots 100 uL cells to student wells, so the OD in the well will be half of the OD in the culture.

100 µL in 96 wells on 2 plates = ~20 mL of cells

Run at RT to be comparable to growth curves (set thermostat to 25C).

Use Script Mode on the plate reader. Program is called E_coli_glow&grow.

Check layout - make sure all rows used are read!

Fix file names so OD, FL, ps1, ps2 are obvious.

Move all the files into a folder labeled with the lab day as soon as the program is through running - otherwise, the data files for the second lab day are interleaved with those for the first lab day. (Sorting by time created may help you out of this fix, though.)

The 12 minute pause is to make the cycle take 15 minutes. Check that lab manual explains this. Enter the time on the compiled file in 15 minute intervals.

2013 - run program for 12 hours. long enough to get past stasis, and show that fluorescence continues to increase.

Give each group of 4 5 mL of each medium

Let them mix in microfuge tubes if they want.

NOTE: plate lid got stuck on PS2 - data completely worthless. Try grinding down the bottom, different lids, tape. Do a dummy run in the morning.

Had to raise up the detector by putting slices of blue ruler under its frame. Also tape the lid to the plate, and the plate to the tray.

LB + Kanamycin (50 µg/mL)
Aliquots in freezer are labeled in 1, 3, 6, 10 mg/mL

If students loaded a whole row with a single solution, they would need 1.2 mL.

antibiotic calculator

Aliquot ~1.5 mL of each medium:

Totals for both days (25 groups)= 37.5 mL

Make 50 mL each
(If there are any left over from the previous year, make ~35 mL, because that is how much can be sterilized in one go with the 35 mL syringe + filter.)

Adding Kanamycin to __mL medium

Sugar solutions

Filter sterilize! Do not autoclave media with sugars!
* lactose takes a long time to go into solution. heat and stir overnight, covered with parafilm.

Strain RP437 - wildtype for chemotaxis

At least 2 days before lab:

Streak all chemotaxis strains on LB agar.

Night before lab

Pick a single colony from the RP437 plate, inoculate into ~5 mL minimal medium + HMLTT; shake at 30C

Morning of lab

Inoculate ~200µL into ~45 mL minimal medium + HMLTT

At class time, OD should be ~0.02

Don't let them reach stationary phase!

To make movies with microscopes in 360:

Frames: 50
Interval: 0.1 sec
exposure: 16.38 msec
pixel type: 8 bit
binning: 4

use x4 list (mark 1) stage position list

shutter open

Slides

coverslips

perfusion chambers (Coverwell 622503 from Grace Bio-Labs) - clean with distilled water

Microscopes

lens paper

NOTE: The optics are bad with the plastic coverslip on the perfusion chamber. If you peel the silicone gasket off the plastic coverslip and replace it with a glass coverslip, you can see much better! (See if there are thinner gaskets.)

Double-sided tape! Put 4 strips of double sided tape on a slide, lower a coverslip over them, and voila! chambers for bacteria! Make sure the tape strips are longer than the coverslip is wide, so you can introduce cells or media without mixing them.

Try double-sided tape with a liner so it is easier to cut and handle, e.g. MMM4010 at WBMason. Scotch Double-Sided Mounting Tape, Industrial Strength, 1" x 60", Clear/Red Liner

For phase to work, the condenser must be at maximum height!

Phase rings must be aligned! Telescope in microscope bulb drawer can be used to align rings. Loosen the chrome screws, adjust the ring with the little black wheels, then tighten. Repeat for each ring/objective combination.

Darkfield may work, too, but condenser must be at maximum height as well.

Minimal Medium + HMLTT

Check stocks in refrigerator before making any solutions!

Plus, for extra-mile experiments:

• 0.01 M glutamic acid

• pH 5.06 (adjust with acetic acid)

• 0.01 M galactose

• 1mM CoCl2

Minimal medium + HMLTT.

Enough to grow overnight cultures and make dilute early log phase cultures of all 7 chemotaxis strains for use during lab.

Swarm Agar

• Mix tryptone and NaCl in water first.

• Bring to final volume.

• Divide into volumes suitable for the vessel(s) going into the autoclave. (E.g., 750 mL per 1 Liter bottle.)

• Add correct amount of agar (3 g per L) to each container. Leave stir bar in container!

• Autoclave 60 min

• Sit on stir plate, stirring, until handleable

• Pour 25 mL per plate. Plates do not keep well, so pour them the morning of the day they will be inoculated.

4 plates per group. (= 48 minimum) So make 1500 mL. Really need 55 plates + some tiny petri dishes to demonstrate swarm agar consistency.

Inoculate at 7 pm: 13 WT plates + 7 of each of the other strains.

Test all vials of frozen strains, especially (or only, depending on the time available) 4130. The strain used F11 grew very poorly. I had to grow them up in LB, then spin them down and resuspend in motility medium.

Try creating a slide with minimal agar + aspartate, etc.

Put a drop of hot agar on a slide, smoosh with a coverslip, add cells around the perimeter.

Do the cells accumulate near the agar? Compare blind and non-blind cells.

Try larger volumes of cells - maybe 6 mL in round bottom tubes.

Try in LB again - maybe fluorescence isn't as bad as we thought originally.