Agarose pads for imaging bacteria

Mon, 01 Jan 0001 00:00:00 +0000

Agarose pads are a simple way to hold bacteria still on a coverslip for microscopy while giving them nutrients from the pad.

Video protocol: how to make agarose pads for imaging happy bacteria under the microscope: .

What it works well for

Tracking bacterial growth. Cells stay in place and keep growing and dividing on the pad. Oxygen is likely limited under the pad, though, so for aerobic bacteria expect up to ~7-8 divisions before growth halts.
Bacterial monolayers for the microscopic phage adsorption assay. This method works amazingly for making a flat, single layer of bacteria (see this in the Gallery). For the assay itself, see . Quick steps for adopting agarose pad protocol for this assay:
- Make 1.5% agarose, keep molten at >45°C.
- Make agarose pad in a cavity slide (as demonstrated in the ).
- Put 2 μL drop of concentrated cells (grown culture concentrated by centrifugation).
- Let the drop visibly dry (~12-15 min at room temperature recommended; 37°C speeds up evaporation but also evaporates too much water from the agarose pad).
- Add 1 μL of phages– concentration pre-determined & adjusted by visualizing labeled phages under the microscope, such that they are sparse enough under the microscope to distinguish the trajectories from one another, but dense enough to yield several tens of phages within the field of view.
- Immediately drop a coverslip on top, seal it on all four sides with VALAP (equal parts VAseline, LAnolin, & Paraffin wax), and start imaging.
- Look for an area on the slide where bacteria are not diffusing in liquid (while the phages are) and there is a nice monolayer: ideally, every pixel covered by cells, something like the region highlighted in yellow below. This is where you want to record the interactions.

Why agarose pads, and not what the paper describes?

In our , we describe tunnel slides made with double-sided tape, with bacteria stuck to the glass by poly-L-lysine. That is what we used to collect the data in the paper.

That approach works well for E. coli, but it is not as good at keeping other bacteria in place: Pseudomonas, for example, keeps diffusing around, only half-stuck. We have since switched to agarose pads, which hold bacteria tigghtly packed together in place (in a monolayer) while giving them ample nutrients, so they stay metabolically active during imaging.

Phage fluorescent labeling

Mon, 01 Jan 0001 00:00:00 +0000

Pass filtered phage lysate (phages removed from bacteria after growth) once through a 100 kDa Amicon filter (in a benchtop centrifuge, not an ultracentrifuge) to exchange the media to PBS (pH 7.4). This helps get rid of molecules in the lysate that the dye might bind to. See my protocol below.

Dye prep

Only done when receiving new dye, not each time labeling occurs.

Fresh 1 mg AZdye TFP ester 488 (or 568 or … you can choose your own based on your filter set) is dissolved into 200 uL water.
10 uL aliquots are made into centrifuge tubes.
Dye is desiccated by running these samples through a speedvac, and the tubes are stored at -20 C in an airtight container with desiccant until further use.

Labeling

A dye aliquot is taken out of the freezer and incubated at room temperature in an airtight container (e.g., a 50 mL centrifuge tube) with desiccant.
Phage lysate (100-500 uL) is dissolved in PBS to a final volume of 4 mL, filtered using 100 kDa Amicon filters, and resuspended in 100 uL PBS.
100 uL of the phage lysate is transferred to the tube with the dye aliquot and pipette-mixed.
The tube is incubated with gentle shaking at room temperature for 2-3 hours. Optional: overnight incubation at 4 C.
Excess dye is washed (through Amicon filters) 4X with PBS.

Phage growth (for beginners)

Mon, 01 Jan 0001 00:00:00 +0000

This protocol is written for folks who are starting out phage work and don’t super care about getting the maximum yield (highest titer or concentration of phages). Once you carry out literature review, you might find facts about the phage of your interest. You may also do some preliminary experiments to determine what works best for your phage.

There are many ways to ~~skin the cat~~ grow your phages; the important thing is that you report the protocol that you ended up using for your published data.

Online protocols and YouTube videos are super helpful. This practical protocol can supplement them, with the hope that each step’s reasoning is explained.

Growth conditions

Growth media & temperature

The rule of thumb is to use the growth media & temperature that you use for growing bacteria. Some phages grow better at certain temperatures, but the physiologically optimum temperature (e.g., 37°C for E. coli) generally works fine for rapid growth.
Ca²⁺ and Mg²⁺ are shown to enhance binding of some phages to hosts. It may increase the phage yield if you add 10 mM MgSO₄ and 5 mM CaCl₂.

Phage stock to propagate

You can…

either start from a previous stock of the phage
or, if you care about picking a single phage genotype for your experiment: make plaques, pick a single plaque, dissolve it in buffer/growth media, and use that solution as the growth source.

Propagation media

There are two main ways to propagate phages: in double-layer agar or in liquid cultures. Both generally work well for lab-adapted phages.

The actual protocol(s)

Propagation in liquid cultures - easier, requires less media prep

Grow bacterial cells to exponential phase in shaking culture flasks.
- Some phages care about the host’s growth phase (e.g., exponential phase may be when maximum number of receptors are expressed), some don’t.
Add 10-100 μL of phages (see “Phage stock to propagate” above).
Grow for 4 h – overnight. Then move on to the isolation step.

Propagation on solid media (Double Layer Agar method)

Bottom agar: 1.5% agar in growth media (similar to agar plates that we pour for growing bacterial colonies)
Top agar: 0.3-0.75% agar in growth media. Keep this molten.
- Autoclaved agar can be stored on the shelf (solidified) and microwaved gradually when used. Gradually means that if you blast the microwave for a few minutes at full power, the bottle will spill which will change effective agar concentration and make a mess in the microwave.
- Exact agar concentration varies from phage to phage. Traditionally common λ or T-phages infecting E. coli grow fine within 0.75% (or lower) top agar. Jumbo phages of Pseudomonas or Enterobacter are temperamental and need low agar concentration (0.3-0.35%) for top agar.
- If you are working with a phage that has published concentration of top agar, just go with what is in the publication. If you got your phage from a collaborator, just ask them what top agar concentration to use.
- When in doubt about not getting phage growth, lower the top agar concentration- this is to account for potential water loss (and hence effective raise in agar concentration) during storage or treatment of the top agar.
Make ~4 mL aliquots of the molten agar and keep them on a heating block around 50°C. Most phages are not degraded at this temperature. If you think that might be happening for you, lower this temperature to ~40-45°C. I would use low-melting agarose instead of agar if lowering this temperature further (to avoid solidification on the heat block which results in chunky top agar on the plate).
Dilute your source phage stock 1:100 (10^-2 dilution) and prepare a 10-fold dilution series (10^-3 – 10^-7).
- Dilution medium can be growth medium (liquid) or salty buffer. I would refrain from using water to avoid osmotic shock for the phages, but have heard of folks using water for robust phages like T4. PBS works great if you are frugal (like me) about restricting access (source of potential contamination) to the growth medium.
- You can dilute 10 μL of original stock in 990 μL for the 10^-2 dilution and 20 μL of previous solution into 180 μL of the dilution medium.
- U-bottom well plates paired with multichannel pipettes are a lifesaver for dilutions, especially if working with multiple phages. If working with just one, microcentrifuge tubes are slightly better (because you can vortex them to mix thoroughly).
Add 100 μL of overnight bacterial culture and 100 μL of a dilution-solution to 4 mL molten agar, vortex the tube to mix the contents, and pour them on pre-warmed bottom agar plates.
- Pre-warming is to avoid immediate and uneven solidification of top agar. Room temperature plates are generally fine, but make sure to pre-warm plates if pulling them out of the fridge.
- Bottom agar is often optional, but helps support robust growth.
Incubate at the desired temperature overnight/longer.
The plates will have varying number of plaques (clearings made by the phage on the bacterial lawn), generally depending on the dilution-solution used. Pick a plate with lacey or webby pattern (plaques overlapping with one another) for phage isolation.
- If plaques are too far apart (in a plate made from a higher dilution of phages), the number of phages isolated will be small. If phage concentration is too high, they will kill all the bacteria and will run out of food source to replicate. So we pick an intermediate dilution to optimize the phage concentration that we get at the end of incubation.
Scoop up the top agar using a spatula, dissolve it in equal amount of liquid (4 mL media or salty buffer), and vortex thoroughly to dislodge phage particles from the agar.

Phage isolation

[Optional] Add a few drops of chloroform to lyse cells and extract any assembled phage particles from within the cells.
- Chloroform will inactivate enveloped/lipid-containing phages (e.g., phage phi6 infecting Pseudomonas syringae), so it should be skipped for those systems.
Centrifuge for 10-15 minutes (anything between 1500×g and 5000×g generally works great) to pellet down cells and/or agar debris.
Pass the supernatant through 0.22 μm filter (PVDF filter works great).
- You might have to use multiple filters, generally when the phage concentration is too high. This is a first-world problem, enjoy your high phage concentration!
- If working with unusually large phages, consider 0.45 μm filters.
The filtrate is your phage solution (also called a “lysate” or “phage stock”).

Storage

Short/medium-term (4°C): Most lab-adapted phages are perfectly happy sitting in the fridge at 4°C, often for months to years. This is the easiest and most common storage method, and is fine for day-to-day lab use.
- Phage titer (concentration) can slowly decay over time at 4°C. The rate of decay depends heavily on the phage. If you’re relying on an old stock for an important experiment, it’s good practice to re-titer it (count plaques again) rather than assume the original titer still holds.
- Store stocks in a buffer or media you trust won’t support bacterial growth if contaminated (e.g., SM buffer or PBS), and keep lysates in tightly capped tubes to avoid evaporation (which concentrates salts/agar and can stress phages).
Long-term (-80°C): For archival stocks (e.g., a reference stock of an important phage you don’t want to lose), freezing at -80°C is common.
- Glycerol (typically 15-25% final concentration) is often added as a cryoprotectant, similar to how you’d freeze a bacterial stock; though many robust phages survive freezing without it. If you’re unsure, check what others have published for your phage of interest.
- Avoid repeated freeze-thaw cycles, which can reduce titer over time. If you expect to use a frozen stock often, consider making several single-use aliquots up front rather than freeze-thawing one tube repeatedly.
- Note that -80°C storage isn’t strictly necessary for most lab-adapted phages. 4°C stocks will likely outlive your PhD. Freezing is more about long-term insurance (e.g., protecting against fridge failure, contamination, or just wanting a “clean” backup you don’t touch).

Bonus: phage enumeration

Follow the dilution series approach mentioned under Propagation on solid media (Double Layer Agar method) above, for 10^-4–10^-9 dilution.
Identify a plate with countable (30–300) plaques.
Count the number of plaques and back-calculate the dilution, like the example below:
- Assume you found 63 plaques at 10^-7 dilution.
- This means 63 PFU (Plaque-Forming Units) were there in 100 uL of this dilution.
- Which implies that there must be 630 PFU in 1 mL of this dilution.
- Hence, there must be 630×10⁷ PFU/mL = 6.3×10⁹ PFU/mL in the original stock.

Practical Protocols | Jyot Antani, PhD

Agarose pads for imaging bacteria

What it works well for

Why agarose pads, and not what the paper describes?

Phage fluorescent labeling

Dye prep

Labeling

Phage growth (for beginners)

Growth conditions

Growth media & temperature

Phage stock to propagate

Propagation media

The actual protocol(s)

Propagation in liquid cultures - easier, requires less media prep

Propagation on solid media (Double Layer Agar method)

Phage isolation

Storage

Bonus: phage enumeration