Using Mycobacterium smegmatis as a Bioindicator for Zinc-Limited Growth Conditions in Mycobacteria

Summary

This protocol details preparation of Zn²⁺-limited media, growth vessels, and seed cultures to produce Zn²⁺-limited ribosomes in mycobacteria. Traces of Zn²⁺ lead to Zn²⁺-limiting growth and we describe morphogenic features used as bioindicators to verify Zn²⁺ limitation in vitro in the model bacterium, Mycobacterium smegmatis, and the human pathogen M. tuberculosis.

Abstract

Many bacteria build alternative ribosomes in Zn²⁺-limiting growth conditions by replacing Zn²⁺-binding ribosomal proteins with Zn²⁺-independent paralogs. Defining a system to study these alternative ribosomes has proven difficult because Zn²⁺ contamination in the laboratory is common. To address this issue, chelating agents are sometimes added to growth media, but this approach convolutes the biological response to gradual Zn²⁺ limitation and is associated with ribosome hibernation. Here, detailed instructions are outlined for preparing media and seeding cultures for Zn²⁺-limited growth without adding chelators. Following this method, the model bacterium, Mycobacterium smegmatis, undergoes morphogenesis, which depends on alternative ribosomes. Because morphogenesis is tractable and only occurs in Zn²⁺-limiting conditions, M. smegmatis can be used as a bioindicator to verify biologically relevant growth conditions. Three bioindicator phenotypes (cell density, cell length, and coenzyme F₄₂₀ fluorescence) that indicate Zn²⁺ limitation in the wild-type are described, and changes in these bioindicators for a deletion mutant that cannot build alternative ribosomes are outlined. Since trace Zn²⁺ contamination is difficult to control for each batch of media, and precise quantification of Zn²⁺ in each media preparation is overly burdensome, following this bioindicator phenotype is an accessible way to validate the preparation of Zn²⁺-limited growth media. To help identify proper conditions for Zn²⁺-limiting growth and alternative ribosome production, changes in the bioindicator phenotypes were profiled for Zn²⁺-contaminated or severely Zn²⁺-depleted preparations of Zn²⁺-limited media as well. Further details to achieve Zn²⁺-limiting growth and alternative ribosome production in M. tuberculosis are presented, along with the associated bioindicator phenotype. Overall, the detailed instructions and bioindicator phenotypes described here will help standardize the production of translationally active alternative ribosomes in mycobacteria.

Introduction

Zinc ion (Zn²⁺) is an essential micronutrient and a standard component of microbiological media. However, many bacteria endure Zn²⁺ limitation in the environment as Zn²⁺-limiting conditions are widespread; soil¹, marine², and niches within the host³ are often Zn²⁺-limited. As such, most bacteria have evolved mechanisms to overcome Zn²⁺ limitation and preserve cellular function⁴. Systems involved with maintaining Zn²⁺ homeostasis are under tight transcriptional control⁵ and are involved in scavenging Zn²⁺ using high-affinity Zn²⁺ import systems, mobilizing cellular Zn²⁺ reserves, and replacing certain Zn²⁺-binding proteins with alternative Zn²⁺-independent paralogs⁶. An example of the latter is Zn²⁺-independent ribosomal protein (RPs) paralogs, which are a common feature of Zn²⁺-responsive regulons in bacteria⁴. These highly divergent Zn²⁺-independent RPs are distinguished from their Zn²⁺-binding paralogs by the disruption of the Zn²⁺-binding CXXC motif⁷. More than half of sequenced prokaryotic genomes contain at least one pair of duplicated RPs where, in many of them, one RP binds Zn²⁺, while its paralog does not⁸. While the C+/C- notation (indicating the presence/absence of the cysteine-rich Zn²⁺-binding motif) is commonly used in reference to these paralogous RP pairs⁷, Zn²⁺-independent RPs have also been described as alternative (Alt) versions of the primary (Prim), Zn²⁺-binding RP paralogs, since AltRPs serve as alternative RPs for ribosome biogenesis in the Zn²⁺-limited condition in some bacteria and it is a keyword with better searchability^9,10,11.

Because AltRP expression is tightly regulated by Zn²⁺, it is logical to assume that AltRP expression offers functional redundancy to Zn²⁺-dependent ribosomal proteins in the face of Zn²⁺ limitation. Indeed, this feature of AltRPs has been demonstrated for the model soil bacterium Bacillus subtilis, where growth is rescued during Zn²⁺ limitation by liberation of the surface PrimRP L31 and replacement with the AltRP L31^12,13. However, some AltRPs are paralogous to core PrimRPs, e.g., S14, meaning their incorporation is required during ribosome biogenesis^12,13. Rebuilding the ribosome with AltRPs is a committed, energy-intensive program. While these core AltRPs may serve to liberate Zn²⁺ from the ribosome, it is also possible that some AltRPs evolved an additional function in the Zn²⁺-limited environment. This is supported by the observation that AltRPs are divergent from their paralogous PrimRPs; sequence homology of AltRP genes is often higher amongst homologs in different species than to the Zn²⁺-binding PrimRP gene sequence within the same species^7,10. Because genes encoding highly divergent AltRPs are under Zn²⁺-dependent transcriptional control, they are removed from the selective pressure exerted on the tightly regulated and highly conserved ribosomal superoperons¹⁴. Decoupled selective pressure on the genetic information encoding the ribosomal machinery and AltRPs exposes these RPs to different evolutionary trajectories than the homologous PrimRPs and could allow for lineage-specific evolution of the same AltRP in different bacteria^10,15. The widespread existence of AltRPs in bacteria and the decoupled genetic regulation of these unique RPs raises an interesting question as to whether the incorporation of AltRPs into bacterial ribosomes confers a special property to AltRP-containing ribosomes (Alt-ribosomes).

Mycobacteria are an ideal group of bacteria to investigate the mechanistic properties of Alt-ribosomes. First, mycobacteria possess a highly conserved altRP operon containing four genes that encode AltRPs: S14-2, S18-2, L28-2, and L33-2^8,9. Mycobacterial AltRPs are significantly different from their PrimRP paralogs; in all four PrimRP/AltRP pairs, a given AltRP gene is more similar across all mycobacterial species than it is to the PrimRP gene in the same species¹⁰. Lineage-specific evolution of AltRPs is also apparent within mycobacteria, as evidenced by AltRPs S18-2 and L28-2 in Mycobacterium tuberculosis and M. bovis, which contain unique sequences at the C-terminus which are not present in other mycobacteria¹⁰. Second, this group of highly related bacteria contain non-pathogenic and pathogenic members that reside in diverse environments from soil (e.g., M. smegmatis) to myriad hosts: human (e.g., M. tuberculosis), bovine (e.g., M. bovis), aquatic (e.g., M. marinum), and avian (e.g., M. avium), to name a few. The highly conserved nature of the altRP operon in mycobacteria, the lineage-specific evolution of specific AltRP genes, and the vastly different environments in which members of this group exist provide a unique opportunity to parse the conserved and lineage-specific evolution of AltRP function and mechanistic properties of Alt-ribosomes. Despite the abundance of AltRPs in prokaryotic genomes, the role of AltRPs and Alt-ribosomes have been investigated in only a few bacteria^{9,10,11,12,13,16,17,18,19}. Establishing a standardized system with specific metrics to validate Zn²⁺-limiting growth conditions in mycobacteria would improve experimental outcomes and advance the understanding of Alt-ribosomes in the field of ribosome research.

Zn²⁺ is a micronutrient; therefore, traces of contaminating Zn²⁺ in chemicals and water contribute to the bioavailability of Zn²⁺, even when omitted from the media preparation. The trace amounts of Zn²⁺ present in water or chemicals can vary drastically between laboratories, thereby affecting the outcome of Zn²⁺-limiting growth. Empirically quantifying the [Zn²⁺] in each independent preparation of media is overly burdensome for most laboratories conducting routine growth assays, but a mechanism to eliminate the possibility of Zn²⁺ contamination in growth media would be intrinsically valuable. To this end, presented here is a detailed method to prepare a chemically defined Zn²⁺-limited media (ZLM), growth vessels, and seed cultures for Zn²⁺-limiting growth and Alt-ribosome production in M. smegmatis and M. tuberculosis. Key morphogenic features (cell density measured by OD₆₀₀, cell length, and fluorescence of coenzyme F₄₂₀) after growth in ZLM are used to verify that Zn²⁺ limitation was achieved. Coenzyme F₄₂₀ is a deazaflavin cofactor that has important roles in catabolism and defense against oxidative stress in methanogens and mycobacteria^20,21. Importantly, mycobacteria are autofluorescent when excited at 420 nm due to the abundance of this cofactor²². The molecular linkage between [Zn²⁺] and F₄₂₀ fluorescence remains unresolved, but following F₄₂₀ fluorescence in cultures is a tractable and dependable bioindicator.

In M. smegmatis, the bioindicator phenotypes occur in the wild-type bacteria, but a deletion mutant lacking the altRP operon does not undergo the same morphogenic program and can be used as a differential bioindicator¹⁰. Although M. tuberculosis does not follow the same morphogenic program as M. smegmatis during Zn²⁺ limitation, M. tuberculosis has a robust and global response to Zn²⁺ limitation and a key bioindicator phenotype following this method²³. Thus, the system defined here enables Prim- and Alt- ribosomes to be generated under the same growth conditions (i.e., Zn²⁺ limitation), facilitating the mechanistic dissection of Alt-ribosome function and its role in shaping bacterial physiology. This growth method was used to resolve the structure of active Alt-ribosomes and demonstrate their translational activity in wild-type M. smegmatis²⁴. Further supporting that this growth method and bioindicator phenotype highlights the biological functionality of AltRPs during Zn²⁺ limitation, AltRP expression is required for the Zn²⁺-dependent morphogenesis and has a profound effect on the transcriptome and proteome of Zn²⁺-limited M. smegmatis^10,25. The same method, with minor modifications in the amount of inoculum and incubation time, is used to generate Zn²⁺-limited cultures of M. tuberculosis^9,23. Here, protocols and bioindicator phenotypes for achieving Zn²⁺-limiting growth in both M. smegmatis and M. tuberculosis are detailed, with a focus on M. smegmatis since this bacterium is more sensitive to Zn²⁺ contamination in the media and is more widely accessible than the slow-growing pathogen.

Protocol

NOTE: The strains used in this study are wild-type M. smegmatis mc² 155 (gift from Dr. Robert Husson, Boston Children's Hospital) and the ΔaltRP deletion strain (originated from Prisic lab¹⁰). They can be cultured in a Biosafety Level 1 (BSL1) or a BSL2 laboratory, depending on local biosafety regulations and available resources. Here, the protocol is written for a BSL2 laboratory. If performed in a BSL1 laboratory, an aseptic technique with a Bunsen burner can replace a biosafety cabinet. In addition, researchers who wish to include other strains or conditions will need to calculate the volume of media needed depending on the total number of cultures assayed. Manipulate pathogenic M. tuberculosis H37Rv (Mtb-H37Rv; gift from Dr. Robert Husson, Boston Children's Hospital) in a BSL3 laboratory wearing a respirator (e.g., PAPR or N95). Do not reuse flasks when culturing highly virulent mycobacteria, such as Mtb-H37Rv. Auxotrophic M. tuberculosis mc²6206 (Mtb-Aux) is attenuated and can be manipulated in a BSL2 laboratory (gift from Dr. William R. Jacobs Jr., Albert Einstein College of Medicine).

1. Preparation of flasks

NOTE: Zn²⁺-limiting growth conditions are achieved in reusable (autoclavable) polycarbonate 250 mL Erlenmeyer flasks. Do not use glass flasks for Zn²⁺-limiting growth or investigate and compare stringent acid-washing procedures to the results presented in this protocol¹⁹. Brand new flasks are adequate to achieve Zn²⁺-limiting growth conditions, but never mix and match new and old flasks in an experiment. Discard flasks that show signs of weakness (usually at the seams) or have been used more than about 20 times. Check cap filters regularly for degradation and dispose if compromised. If present, discard the cap lids that screw onto the top of the caps and cover the filters. This method outlines the precautions taken to clean the flasks and prepare them for reuse.

1. Permanently label the flasks so the same set is only used for Zn²⁺-limiting conditions. Label another set of flasks for Zn²⁺-replete growth. Labeling with different colors is useful to prevent mixing the flasks.
2. Never allow culture residue to dry in the flasks. Add water to the flasks immediately after decanting the culture from the flask (e.g., 100 mL). If the culture is not harvested at the end of the growth, empty the flasks by pouring the entire culture into a liquid waste disposal receptacle with 10% final bleach concentration after recording the bioindicator phenotypes as described in this protocol.
3. Autoclave flasks on liquid cycle (at least 121 °C at 15 psi) for 20 min with the water inside and the lids on.
4. After autoclaving, wash flasks with nonionic, low-foaming detergent and scrub well using a soft scrubber brush. Dedicate a brush to be used only for washing flasks used for Zn²⁺-limiting growth. Ensure flasks are clean with no residue or debris. Do not scrub or use soap on caps, rinse gently with deionized water.
5. Rinse the flasks well with tap water, followed by three rinses with deionized (type II or III) water and at least one more rinse with ultrapure (type I) water (18.2 MΩ-cm at 25 °C and ≤ 5 ppb of total organic carbon).
6. Place the flasks vertically upside down so no water can pool in the bottom. Do not use standard wall-mounted laboratory drying racks, as these hold flasks at an angle and allow a small pool of water to collect. Standard dishware racks will suffice. Let the flasks and the lids dry completely.
7. Once dry, screw the caps onto the flasks and cover the lids with aluminum foil. Affix a piece of autoclave indicator tape to the foil to indicate sterile status and to protect the cap filters from dust.
8. Autoclave the cleaned flasks (at least 121 °C at 15 psi) for 20 min, followed by a post-cycle vacuum to dry the load (if the autoclave is equipped with this feature). Store the flasks dry and protected from dust.

2. Preparation of Zn²⁺-limited medium (ZLM)

NOTE: Use only plastic beakers, graduated cylinders, and any other supplies to prepare media. Designate a set of labware that is used exclusively for the preparation of Zn²⁺-limited media. Rinse all labware thoroughly with ultrapure (type I) water before and after use. Use chemicals of high purity and buy from the same suppliers, if possible.

1. Prepare Zn²⁺-limited Sauton's medium without added Zn²⁺ salts²⁶ by mixing the following chemicals with ~950 mL of ultrapure water in a 1 L plastic beaker with a stir bar:
   0.5 g KH₂PO₄ (0.05%; MW: 136.09)
   0.5 g MgSO₄ x 7H₂O (0.05%; MW: 246.48)
   2 g citric acid, anhydrous (0.2%; MW: 192.12)
   4 g L-asparagine, 99% (0.4%; MW: 132.12)
   60 mL glycerol (6%)
   189 µL1 M ferric ammonium citrate (0.005%; MW: 265)
   1. Add glycerol using a disposable, plastic 50 mL serological pipette. Rinse the pipette out well by pipetting up and down about 10x in the medium until no residual glycerol remains in it.
      NOTE: Glycerol is highly viscous and difficult to measure. Alternatively, glycerol can also be poured into a graduated cylinder and rinsed out numerous times with about 30 mL of ultrapure water that is then added to the media. If taking this approach, start with less volume (~600 mL) so the final volume is not greater than ~950 mL after adding the glycerol and washes from the graduated cylinder to the media.
2. Bring up the volume of the media to about 950 mL and mix well. The L-asparagine takes a few minutes to go into solution; wait until there are no white flakes remaining before proceeding.
   NOTE: The ferric ammonium citrate solution makes the media a light-yellow color.
3. Prepare 15 mL of fresh NaOH solution (~10% w/v) with ultrapure water in a plastic container (e.g., 15 mL conical tube).
4. Thoroughly rinse a pH meter with ultrapure water before use and immerse only the bulb of the pH meter into the media. Leave the pH meter in the medium with gentle stirring while adjusting the pH. Using a plastic disposable transfer pipette, add the freshly prepared NaOH to a final pH of 7.4.
   NOTE: The starting pH of the media is highly acidic. It takes a large volume of NaOH solution to bring the pH up to the neutral range. Add 4-6 mL of the NaOH solution initially; once the pH reaches around 6.5, add the NaOH solution dropwise. Do not overshoot the target pH of 7.4. If this occurs, do not try to re-adjust with HCl or other acids - discard the media. It is good practice to dedicate a pH meter for making ZLM and not for measuring the pH of other media and buffers that could contaminate the probe.
5. Transfer the media to a graduated cylinder and bring it up to a final volume of 1 L with ultrapure water. Filter sterilize the media through a 0.2 µm disposable filter flask.
6. Aseptically add 2.5 mL of filter-sterilized 20% Tween-80 solution (0.05% final concentration). Store at 4 °C away from light for up to 6 months.

3. Zn²⁺-limiting growth in M. smegmatis

NOTE: Due to batch variation in the amount of Zn²⁺ present in ZLM, it is imperative to use the same batch of media or multiple media that have been well mixed together for all cultures in an experiment. In addition to the media required to set up cultures, about 100 mL extra is needed for washing and normalizing the seed cultures.

1. Perform all steps in a biosafety cabinet. Prepare seed cultures 18 h prior to setting up a growth experiment. Do not use overgrown or clumping seed cultures. One seed culture per strain is sufficient.
   1. Aseptically add 5 mL of 7H9-ADC medium (Middlebrook 7H9 broth with 0.05% Tween 80 and 10% ADC enrichment [final concentration: 0.5% bovine serum albumin, 0.2% dextrose, 0.085% NaCl and optional 0.0003% catalase]) to a 50 mL bioreactor tube.
   2. Aseptically add 10 µL of thawed glycerol stock cells (prepared according to²⁶) directly to the medium. Screw the lid onto the tube.
   3. Position the tubes firmly in a shaking incubator at approximately a 45° angle so the liquid goes about halfway up the tube while shaking.
   4. Incubate tubes at 37 °C with shaking at 120 rpm for approximately 18 h.
2. Check overnight seed cultures to ensure the culture is evenly turbid and is not significantly clumping. The optical density at 600 nm (OD₆₀₀) of the overnight culture is ideally around 0.5-0.8.
3. Pellet overnight cultures in the bioreactor tubes, used for overnight growth, by centrifugation at 3,000 x g for 5 min at room temperature.
4. Decant or aspirate the supernatant and resuspend in 5 mL of ZLM. Repeat the centrifugation and resuspension steps 2x for a total of three washes.
   1. During centrifugation steps, fill a cuvette until overflowing with 70% ethanol to sterilize. Allow the filled cuvette to sit in the biosafety cabinet for 10 min and rinse well 3x with ZLM using a pipette.
      NOTE: The cell normalization process takes time and practice to achieve efficiently. By normalizing the cells directly in the ethanol-sterilized cuvette, the process is greatly accelerated.
   2. Prepare enough cuvettes for the number of strains included in the study, e.g., this protocol requires two sterilized cuvettes, one for wild-type and one for the ΔaltRP mutant.
5. After three washes with ZLM, resuspend each cell pellet in 2 mL of ZLM.
6. Blank a spectrophotometer with 600 µL of ZLM in a cuvette. The cuvette does not need to be sterilized. Set aside the cuvette and use it to blank the spectrophotometer before normalizing each culture (i.e., strain).
7. Working with one strain at a time, transfer 600 µL of washed cells from step 3.5 to a sterilized cuvette prepared in step 3.4.1 and measure the starting OD₆₀₀. If the OD₆₀₀ is above 1, proceed to step 3.8. If the OD₆₀₀ is below 1, follow the sub-steps 3.7.1 and 3.7.2.
   1. If the starting OD₆₀₀ reading from step 3.7 is below 1, collect all the culture from within the cuvette, add it back to the remaining washed seed cells in the centrifuge tube, and centrifuge the culture again as in step 3.3. Resuspend the culture in a lower volume than was used in step 3.5 (e.g., 1 mL) to achieve a higher cell density, and repeat step 3.7.
   2. If the OD₆₀₀ after step 3.7.1 is still below 1, the culture is not dense enough; restart the procedure from step 3.1. Incubate cultures longer or increase the amount of inoculum for setting up the overnight cultures such that the final overnight culture density is around OD₆₀₀ = 0.5-0.8, as described in step 3.2.
8. Calculate the volume of ZLM to be added to the 600 µL of washed cells in the cuvette to obtain an OD₆₀₀ of 1 using the following equation and the OD₆₀₀ measured in step 3.7:
   Volume of ZLM (μL) = (starting OD₆₀₀ - 1) × 600 μL
9. Add the volume of ZLM calculated in step 3.8 to the cuvette prepared in step 3.7, mix well by pipetting 5x-10x, and re-measure the OD₆₀₀.
10. Adjust the OD₆₀₀ to within 5% of OD₆₀₀ = 1 following the sub-steps listed below. Use two pipettes where one (P1000 size) is dedicated to mixing the culture to avoid using too many tips for each adjustment made.
    1. If the OD₆₀₀ is above 1, add ZLM dropwise (~50 µL at a time), mix well by pipetting up and down 5-10 times, and measure the OD₆₀₀. Repeat this process as needed until the culture is within 5% of OD₆₀₀ = 1.
    2. If the OD₆₀₀ falls below 1 during normalization, add more washed cells (~20 µL to begin) from step 3.5, mix well by pipetting up and down 5x-10x, and measure the OD₆₀₀. Repeat this process as needed (or add more ZLM as described in step 3.10.1 if the OD₆₀₀ goes above 1) until the culture is within 5% of OD₆₀₀ = 1.
    3. Ensure the volume in the cuvette does not exceed ~1.5 mL during cell normalization to avoid spilling culture in the spectrophotometer. Remove culture from the cuvette when diluting, if needed. Also ensure the culture volume in the cuvette is above the minimum required to accurately measure OD₆₀₀ for the spectrophotometer. Most spectrophotometers require at least 600 µL.
11. Repeat the cell normalization steps (3.6 - 3.10) for all strains and conditions included in the study.
12. Prepare the total number of flasks required for an experiment by aseptically adding 50 mL of ZLM into 250 mL plastic Erlenmeyer flasks prepared as described in step 1.
13. Inoculate flasks by aseptically adding 50 µL of the OD₆₀₀ = 1 normalized culture directly from the cuvette for a final OD₆₀₀ of 0.001 (1:1000 dilution of normalized inoculum).
14. For cultures that are grown without Zn²⁺ limitation (i.e., Zn²⁺-replete media, ZRM), add 30 µL of sterile 10 mM ZnSO₄ (prepared fresh from 1M sterile stock solution) directly to the culture flasks to a final concentration of 6 µM ZnSO₄.
15. Incubate flasks at 37 °C with shaking at 100 rpm for 3 days to stationary phase (Day 0 is the day of inoculation).
16. Each day of growth, use a monochromator or filter-based microplate reader to measure the following growth metrics using 200 µL of culture in a 96-well flat-bottom microplate. Take plate readings immediately after aliquoting the cells so they do not settle.
    NOTE: Pre-heating the plate reader is not necessary as the absolute fluorescence values are not as informative as the ratio and fluorescence scans, and the small volume measured rapidly equilibrates to room temperature during plate set-up. Fluorescence can be measured from the top or bottom of the plate if condensation on the lid is problematic, although fluorescence intensity values are overall lower when read from the bottom.
    1. Cell density (i.e., OD₆₀₀): To report the OD₆₀₀ obtained from a microplate reader, generate a standard curve to convert the reading from the microplate path length using a set volume (e.g., 200 µL) to the standard 1 cm path length.
    2. Fluorescence of cofactor F₄₂₀ (ex 420 nm/em 475 nm): For filter-based microplate readers, use the closest filter set (e.g., ex 430 nm/em 488 nm) and black 96-well microplates.
    3. Measure background fluorescence at 375 nm (ex 375 nm/em 475 nm)
    4. If using a monochromator, generate a fluorescence intensity scan from ex 230 - 440 nm in 5 nm steps and fixed emission at 475 nm.
    5. If using a monochromator, calculate the percentage of fluorescence intensity at 375 nm to 420 nm using the equation:
       F_375/420 (%) = (F₃₇₅/F₄₂₀) x 100

4. Recording and measuring cell length

1. On day 3, visualize and record cell length. Clean a glass slide and coverslip with a lint-free wipe. Pipette a 10 µL drop of culture onto the slide and drop the coverslip directly on top of the culture. Gently press the coverslip so the culture spreads and nearly fills the entire area underneath the coverslip. Prepare slides and visualize immediately to prevent drying.
   NOTE: If the culture does not easily spread out, debris prevents good contact, and water movement will obscure capturing cells in focus. Prepare a new slide.
2. Using a compound microscope (or DIC, see below) with 100x oil immersion and 10x ocular objectives (1000x total magnification) and a camera attachment, visualize the prepared slide.
   NOTE: Cells are much easier to see with the contrast from differential interference contrast (DIC) microscopy¹⁰. Use this type of microscope for high quality images, if possible. For routine analysis and verification of Zn²⁺-limited growth status, a standard compound light microscope is sufficient.
3. Take pictures of areas of the slide with multiple cells that are in focus, in plane (i.e., the entire length of the cell is visible) and are not moving. Record enough pictures throughout different areas on the slide (or across multiple slides of the same culture) so that at least 100 cells are measured.
4. Using the same microscope and objective, image a calibration slide.
5. In Fiji²⁷, use the straight-line tool to draw a line across the length of the calibration slide. Select Analyze > Set scale and fill in the known distance and unit of the scale bar. Select Global to apply the scale to all future images and measurements.
6. Open a microscope photo. Use the straight-line tool to draw a line across the length of a cell and press the M key on the keyboard to measure the cell. Measure at least 100 cells of each culture.
7. Copy length data generated in the pop-up window in Fiji into a spreadsheet software or save it as a comma-separated value (.csv) file for statistical analysis.

5. Modifications for growing Zn²⁺-limited M. tuberculosis (Mtb)

NOTE: In contrast to Mtb-H37Rv, Mtb-Aux cannot synthesize L-leucine and D-pantothenate, and these supplements are added to the media²⁸. The supplementation for Mtb-Aux does not affect Zn²⁺ limitation, and both strains display similar phenotypes in ZLM²³. This protocol is modified from previously published protocol²³, the main difference being that cultures are grown for 8 days instead of 10 days to decrease clumping and allow for enrichment for single cells, which is needed for downstream applications such as macrophage infections. For other minor differences, please refer to the original publication²³. The method of inoculation differs from M. smegmatis due to the much higher inoculum needed to initiate the growth of M. tuberculosis.

1. For Mtb-Aux, provide additional supplementation of 50 µg/mL L-leucine and 48 µg/mL D-pantothenate in all media. Filter sterilize 1000x stock solutions of 50 mg/mL L-leucine and 48 mg/mL calcium D-pantothenate with a 0.2 µm filter and add to sterilized media.
2. Aseptically add 1 mL of thawed glycerol stock cells to 4 mL of 7H9-ADC in a 14 mL round bottom snap-cap tube (for Mtb-Aux) or a 50 mL bioreactor tube (for Mtb-H37Rv).
3. Incubate at 37 °C without shaking until the culture reaches OD₆₀₀ ~1. This will take about 1 week. Aerate the culture once a day by opening the tube in a biosafety cabinet and pipetting a few times to mix the culture.
4. Inoculate 1 mL of the standing culture into 50 mL of 7H9-ADC in a 250 mL vented plastic flask. One 50 mL culture in 7H9-ADC is enough for biological triplicates per strain and condition (i.e., wild-type in ZLM and ZRM). Take care to avoid large clumps (i.e., large aggregates of cells) by letting the culture stand for 5-10 min prior to inoculation, allowing clumps to settle to the bottom of the tube, and taking the culture from the top.
   NOTE: Inoculation with large clumps yields clumpy cultures, which affects culture normalization and reproducibility.
5. Incubate at 37 °C with shaking at 100 rpm until the culture reaches OD₆₀₀ 0.5-0.8 (mid-log phase). This will take 3-5 days.
6. Transfer culture to a 50 mL conical tube and pellet cells by centrifugation at 3,000 x g for 10 min at room temperature. Discard the supernatant by aspirating 40 mL with a serological pipette and removing the remaining supernatant with a P1000 micropipette.
   NOTE: This step is critical to reduce Zn²⁺ carryover from the 7H9-ADC medium.
7. Resuspend the pellet in 5 mL of ZLM and bring up the total culture volume to 50 mL in the conical tube with ZLM.
8. Repeat centrifugation at 3,000 x g for 10 min at room temperature to pellet cells. Discard supernatant. Resuspend the cell pellet to 50 mL final volume as described in step 5.7.
9. Measure the OD₆₀₀ of the washed seed cells and calculate the volume required to obtain a starting OD₆₀₀ of 0.05 (Mtb-H37Rv) or 0.08 (Mtb-Aux) in 50 mL using the following equation:
   
   NOTE: Mtb-Aux growth with a starting OD₆₀₀ of 0.05 is inconsistent; thus, a higher OD₆₀₀ of 0.08 is used for consistent growth.
10. Aliquot the volume of seed culture calculated in step 5.9 into a new 50 mL conical tube and bring up the volume to 50 mL with ZLM. Decant the washed and normalized culture from the conical tube into plastic Erlenmeyer flasks prepared as described in step 1. Repeat using the same conical tube for all biological replicates for each strain and condition.
11. For cultures grown without Zn²⁺ limitation (i.e., Zn²⁺-replete media, ZRM), add 30 µL of sterile 10 mM ZnSO₄ directly to the culture flasks to a final concentration of 6 µM ZnSO₄.
12. Incubate culture flasks at 37 °C with shaking at 100 rpm for 8 days. Measure OD₆₀₀ and F₄₂₀ fluorescence throughout growth as described in step 3. Discard cultures if they are clumpy within the first few days of growth (i.e., the culture does not look turbid, and there are clumps throughout the culture).
    NOTE: Ideally, cultures are mostly planktonic (i.e., free of large clumps that are visible by eye) during the first few days of growth. Mtb cultures, especially ZLM cultures, become progressively clumpier throughout growth, and clumps floating on the surface of the media in flakes are expected by the late log phase²³.

Results

Bioindicator phenotypes of M. smegmatis
Wild-type M. smegmatis cultures grown in ZLM that achieve Zn²⁺-limiting growth have three main bioindicator phenotypes when compared to cells grown under Zn²⁺-replete conditions: [1] decreased OD₆₀₀, [2] elongated cells, and [3] decreased peak fluorescence at 420 nm. The mutant lacking AltRPs (ΔaltRP) does not have the same Zn²⁺-dependent morphogenesis as the wild-type^10, and its unique phenotype in Zn²⁺-limited media can be used as a differential bioindicator. Therefore, all three bioindicator metrics should first be evaluated in wild-type grown in ZRM and ZLM. If the expected Zn²⁺-limited wild-type phenotype is observed, the ΔaltRP deletion strain should then be evaluated. Growth in ZLM does not always achieve Zn²⁺-limited conditions, which is the very reason why bioindicators are needed. Unique wild-type and ΔaltRP bioindicator phenotypes verify that biologically relevant Zn²⁺-limiting conditions were achieved. When cultures grown in ZLM do not achieve Zn²⁺-limiting growth due to Zn²⁺ contamination in the media or culture vessels (ZLM-C), these bioindicator phenotypes will be less pronounced, as shown in Figure 1 and Figure 2. Conversely, if the concentration of Zn²⁺ in ZLM is too low, growth is impaired by Zn²⁺ depletion (ZLM-D), which is evident in low culture turbidity and altered bioindicator phenotypes, also detailed in Figure 1 and Figure 2.

Cell density is a strong metric of Zn²⁺ limitation. Cultures grown in ZRM consistently reach high turbidity (above OD₆₀₀ of 5) after 72 h, maybe clumping, and are visibly more yellow (data not shown) than cultures grown in ZLM (Figure 1A). Zn²⁺ limitation restricts growth (marked by decreased OD₆₀₀) of all strains when compared to the Zn²⁺-replete condition, and the ΔaltRP strain has a lower cell density than the wild-type (Figure 1A). Zn²⁺-limiting growth in ZLM consistently yields an OD₆₀₀ above 3.5 and less than 5 in the wild-type and less than 3 in the ΔaltRP strain (Figure 1A). Cultures grown in ZLM that had Zn²⁺ contamination (ZLM-C) attain a similar cell density (OD₆₀₀ is above 5) to Zn²⁺-replete cultures grown in ZRM (Figure 1A). If the [Zn²⁺] in ZLM is too low, cultures will be severely depleted for Zn²⁺ (ZLM-D), and growth is impaired, as evidenced by decreased cell density in wild-type cultures (OD₆₀₀ is less than 3; Figure 1A). Wild-type cell density (3.5 < OD₆₀₀ < 5) should be the first bioindicator phenotype checkpoint to verify Zn²⁺-limiting conditions. Only wild-type cultures that grow to the expected cell density should be considered, and analysis of the ΔaltRP strain should come second to verifying Zn²⁺-limited bioindicator phenotype in the wild-type.

Stationary phase wild-type cultures grown in ZLM that are limited for Zn²⁺ are elongated with an average cell length of around 9 µm, whereas Zn²⁺-replete cultures grown in ZRM are short with an average cell length of around 3 µm (Figure 1B). Wild-type cultures grown in ZLM with Zn²⁺ contamination (ZLM-C) do not fully elongate but are slightly longer than Zn²⁺-replete cells with an average cell length of 5 µm. Bioindication of Zn²⁺ contamination in whole batches of ZLM indicates possible contamination during preparation, while in individual cultures(i.e., flasks) indicate contamination due to improper washing of flasks or culture preparation. Wild-type cultures grown in ZLM that are severely depleted for Zn²⁺ (ZLM-D) have less-pronounced elongation with an average cell length of around 7 µm (Figure 1B). The elongated wild-type phenotype is not observed in the ΔaltRP strain, and ΔaltRP cells that are limited for Zn²⁺ have an average cell length of around 5 µm (Figure 1B). Figure 2 shows representative images of cells grown in different preparations of ZLM or ZRM (following step 2) and visualized with a compound light microscope. Following step 4, cells from numerous fields in images like what is shown in Figure 2 are used to calculate the average cell length of the cultures shown in Figure 1B. The wild-type elongated phenotype is specific to Zn²⁺-limiting conditions; cellular elongation is not observed when cultures are Zn²⁺-replete or severely Zn²⁺-depleted (Figure 1B and Figure 2).

The decreased F₄₂₀ fluorescence bioindicator phenotype is best observed in fluorescence scans (Figure 1C). Decreased fluorescence from cofactor F₄₂₀ is quantified by taking the percentage of fluorescence intensity at 375 nm (background) to 420 nm (autofluorescence from cofactor F₄₂₀) as described in step 3.16.5, represented as F_375/420. For monochromatic plate readers without fluorescent scanning ability, point readings at 375 nm and 420 nm (both with emission at 475 nm) are suitable to calculate F_375/420. Wild-type cultures grown in Zn²⁺-replete conditions (i.e., in ZRM) have highly reproducible F_375/420 around 17% (Figure 1D). Reduction in F₄₂₀ fluorescence in Zn²⁺-limited cultures grown in ZLM is marked by an increase in the F_375/420 to around 27% (Figure 1D). F₄₂₀ fluorescence in the Zn²⁺-limited ΔaltRP strain is remarkably lower than the wild-type in the same condition, and this is visually evident in fluorescence scans (Figure 1C). The F_375/420 is more variable in the Zn²⁺-limited ΔaltRP strain, with a range from about 50%-80% (Figure 1D). Cultures grown in ZLM but showing bioindication of Zn²⁺ contamination (ZLM-C) have reduced F_375/420 to around 21%, and cultures that are Zn²⁺-depleted (ZLM-D) have F_375/420 to around 35% (Figure 1D).

In summary, three bioindicator phenotypes should be assessed in the wild-type and ΔaltRP strains to verify the Zn²⁺-limited status of cultures grown in ZLM. The bioindicators should be as follows for biologically relevant Zn²⁺-limiting growth: [1] OD₆₀₀ of the wild-type is 3.5 - 5, and the ΔaltRP strain is less than 3, [2] wild-type cells are around 10 µm long, and ΔaltRP cells are half that length, and [3] F_375/420 of the ΔaltRP strain is high, i.e., above 50%.

Bioindicator phenotypes of M. tuberculosis
Zn²⁺-limited M. tuberculosis does not show the same morphogenic program as M. smegmatis. Specifically, there is no difference in the OD₆₀₀ between Zn²⁺-replete and Zn²⁺-limited cultures, and Zn²⁺-limited cultures do not demonstrate any obvious morphological phenotype, i.e., they do not elongate²³. Similarly, there is not a Zn²⁺-depleted bioindicator phenotype (i.e., reduced cell density) in M. tuberculosis as is observed in M. smegmatis. Zn²⁺-limited cultures have more apparent clumping compared to Zn²⁺-replete cultures, though both conditions are considerably clumpy by day 10 (observed in both Mtb-H37Rv and Mtb-Aux). The key bioindicator phenotype for Zn²⁺ limitation in M. tuberculosis is reduced peak F₄₂₀ fluorescence, as previously reported²³. Results for cultures grown 8 days are presented here instead of 10 days to minimize clumping. Decreased F₄₂₀ fluorescence is apparent in Zn²⁺-limited cultures with both monochromatic (Figure 3) and filter-based plate readers. For filter-based plate readers, using Mtb-H37Rv, Zn²⁺-limited cultures (i.e., grown in ZLM) have about 50% of the raw fluorescence values as Zn²⁺-replete cultures (i.e., grown in ZRM; data not shown). Using a monochromatic plate reader, bottom-reading mode, and Mtb-Aux, Zn²⁺-replete cultures have F_375/420 around 75%, and Zn²⁺-contaminated ZLM cultures have similar values. Decreased F₄₂₀ fluorescence in Zn²⁺-limited cultures grown in ZLM is reflected by F_375/420 of over 100%. The Zn²⁺-limited ΔaltRP strain has similar growth and F₄₂₀ fluorescence as seen in the Zn²⁺-limited wild-type (Figure 3) and is not useful as a bioindicator in M. tuberculosis as it is in M. smegmatis (Figure 1). Therefore, it is advisable to test media that will be used to grow M. tuberculosis with the fast-growing M. smegmatis.

Figure 1: Bioindicator phenotypes of M. smegmatis grown under Zn²⁺ limitation and the effect of Zn²⁺ contamination and Zn²⁺ depletion on the phenotypes. Representative examples of the bioindicator phenotype shown in each panel are from the same cultures at 72 h. Examples of Zn²⁺-contaminated (ZLM-C), Zn²⁺-limited (ZLM-L), and Zn²⁺-depleted (ZLM-D) growth outcomes are from independent media preparations that all followed the same method of preparation presented here (step 2). The representative phenotypes for the Zn²⁺-limited wild-type and ΔaltRP strains are from the same batch of ZLM. All strains and conditions have three biological replicates (n=3). (A) Growth curves showing optical density detected at 600 nm (OD₆₀₀) and decreasing cell density with Zn²⁺ limitation and depletion. Error bars represent the standard deviation between replicates. (B) Cell lengths of wild-type and ΔaltRP strains demonstrating the elongated phenotype of Zn²⁺-limited wild-type. Box and whisker plot shows the interquartile distance, minimum, maximum, and median cell lengths in µm. (C) Representative fluorescence scans of cultures showing decreased peak F₄₂₀ fluorescence with Zn²⁺ limitation and diminished F₄₂₀ fluorescence in Zn²⁺-limited ΔaltRP strain. Only one representative culture from the biological triplicates in each of the different media preparations is shown. Fluorescence scans use an excitation wavelength range of 230 nm to 440 nm with a 5 nm step size and an emission wavelength of 475 nm. The in-pane panel shown on the upper left is a zoom-in view of the Zn²⁺-limited wild-type and ΔaltRP strains to accentuate the decreased fluorescence peak at 420 nm in the Zn²⁺-limited ΔaltRP strain. Axis units are the same as the main graph. All legends are the same as in (A). (D) Quantification of decreased fluorescence from cofactor F₄₂₀, calculated from the percentage of F_375/420. Percentages are calculated from the values obtained from the fluorescence scans in (C). Box and whisker plot shows the interquartile distance, minimum, maximum, and median percentages. Abbreviations representing the Zn²⁺-relevant status of the medium provided in parentheses after the media type are R = Zn²⁺-replete, C = Zn²⁺-contaminated, L = Zn²⁺-limited, and D = Zn²⁺-depleted, and other abbreviations are: WT = wild-type strain, a.u. = arbitrary units. Please click here to view a larger version of this figure.

Figure 2: Representative microscopy images of M. smegmatis at 72 h. Pictures are from cultures shown in Figure 1. Cells were imaged using a compound light microscope at 1000x total magnification using a 100x oil immersion lens. Scale bars are 10 µm. Abbreviations representing the Zn²⁺-relevant status of the medium provided in parentheses after the media type are: R = Zn²⁺-replete, C = Zn²⁺-contaminated, L = Zn²⁺-limited, and D = Zn²⁺-depleted, and other abbreviations are: WT = wild-type strain. Please click here to view a larger version of this figure.

Figure 3: Loss of F₄₂₀ bioindicator phenotype in Zn²⁺-limited M. tuberculosis demonstrated by representative fluorescence scans of auxotrophic Mtb mc²6206 (Mtb-Aux). Examples of Zn²⁺-contaminated and Zn²⁺-limited growth outcomes are from independent media preparations. The Zn²⁺-limited wild-type and ΔaltRP strains are from the same batch of ZLM. All strains and conditions have three biological replicates (n=3). Only one representative culture from the biological triplicates in each of the different media preparations and strains is shown in the graph. Fluorescence scans were taken using bottom-reading mode after 8 days of growth using an excitation wavelength range of 230 nm to 440 nm with a 5 nm step size and an emission wavelength of 475 nm. Abbreviations representing the Zn²⁺-relevant status of the medium provided in parentheses after the media type are: R = Zn²⁺-replete, C = Zn²⁺-contaminated, L = Zn²⁺-limited, and D = Zn²⁺-depleted, and other abbreviations are: WT = wild-type strain, a.u. = arbitrary units. Please click here to view a larger version of this figure.

Discussion

A major contribution to the knowledge gap on mechanistic underpinnings of Alt-ribosomes is the difficulty in achieving Zn²⁺-limiting conditions in vitro. Trace Zn²⁺ contamination in the laboratory setting is common; thus, achieving reproducible Zn²⁺-limited growth conditions is challenging. Zn²⁺ contamination can originate from several sources, although the most significant and common source of contamination and variability is from the ultrapure water used to rinse labware and make media. Ultrapure water systems should be properly maintained and should be flushed for 1 min before rinsing labware or using water for media. Flasks that are not washed or rinsed well with ultrapure water may cause Zn²⁺ contamination, and this will be apparent by the increased cell density in certain flask(s) but not others from the same batch of ZLM. Brand new flasks can be used to assess if the source of Zn²⁺ contamination is from reused flasks or media. Zn²⁺ contamination in ZLM should be remediated by increased rinsing of items used to make media and a designated set of materials for ZLM preparation. If none of these measures prove successful in achieving the expected bioindicator phenotypes, the [Zn²⁺] in ultrapure water and ZLM should be quantified to rule out the possibility that the water source is Zn²⁺-contaminated. As previously reported, the [Zn²⁺] of ZLM is 115 ± 37 nM²³, and concentrations below 70 nM and above 150 nM usually led to Zn²⁺-depleted and Zn²⁺-contaminated phenotypes, respectively. However, good media preparation is only one factor that affects the outcome, and inadequate or excessive Zn²⁺ may occur due to the improper preparation of the inoculum and/or culturing, i.e., poor decanting of Zn²⁺-replete media during wash steps. All M. smegmatis cultures included here were prepared in the same way - Zn²⁺-contaminated (ZLM-C) and Zn²⁺-depleted (ZLM-D) growth outcomes are included to highlight the variability that can occur between different batches of media using the same ultrapure water system, even when experiments are done correctly.

To ensure that culturing cells in ZLM yields reproducible Zn²⁺-limiting bioindicator phenotypes, it is important to uniquely label the flasks, always use them for the same condition (i.e., with ZLM or ZRM), track flasks over multiple uses and dispose flasks that are at the end of their lifecycle, or that continue to yield Zn²⁺-contaminated bioindicator phenotypes with ZLM. Proper inoculum preparation is very important to achieve Zn²⁺-limiting growth, as Zn²⁺ carry-over from inadequately washed seed cells is a common source of Zn²⁺-contamination that can be difficult to parse from Zn²⁺-contaminated ZLM. M. smegmatis cultures should be assayed and harvested after 72 h of growth in ZLM (and ZRM grown in parallel). The morphogenesis and bioindicator phenotypes are pronounced from 48-72 h; therefore, evaluating or harvesting cultures for Zn²⁺ limitation too early (or too late) may not yield the full bioindicator phenotype. Cultures that are depleted in Zn²⁺ will exhibit cell lengths that are similar to those that are Zn²⁺-contaminated; thus, F₄₂₀ fluorescence and OD₆₀₀ should be used to differentiate the two. Likewise, Zn²⁺-contaminated cultures may not always grow better than Zn²⁺-limited bacteria, but F₄₂₀ fluorescence and the cell length of the wild type and the ΔaltRP mutant should reveal if the Zn²⁺-limited condition is achieved.

The same is true for M. tuberculosis, as the loss of F₄₂₀ bioindicator phenotype in Zn²⁺-limited cultures is pronounced between 8-10 days of growth in ZLM. Though M. tuberculosis does not exhibit a strong Zn²⁺-limited bioindicator phenotype like M. smegmatis, the growth method presented here has been used to associate loss of F₄₂₀ bioindicator phenotype with Zn²⁺-limited growth using functional assays and transcriptomics in both the virulent and attenuated strains of M. tuberculosis (ref²³and unpublished data). Therefore, following F₄₂₀ fluorescence is an accessible way to monitor and verify biological Zn²⁺-limiting conditions in M. tuberculosis. It is important to note that the mechanism leading to reduced F₄₂₀ fluorescence during Zn²⁺-limiting conditions remains unresolved, as is the distinction of whether the reduced fluorescence is due to decreased abundance of the coenzyme pool as a whole or a shift in its redox state since only the oxidized form emits fluorescence²⁹.

As demonstrated here, the preparation of Zn²⁺-limiting media is nuanced, and there are numerous factors that can influence the effective concentration of Zn²⁺ ions in the media even when following the same protocol and using the same reagents. Traces of Zn²⁺ in ZLM allow for sufficient growth and expression of the Zn²⁺-limiting phenotype. This gradual decrease of Zn²⁺ to achieve limitation is similar to the Wayne model of hypoxia in which oxygen is gradually consumed by bacteria grown in a closed flask³⁰. Since both hypoxia and Zn²⁺-limited phenotypes depend on bacterial growth, if there is not enough oxygen, or in this model, Zn²⁺, in the inoculum or medium, bacteria will stop growing before they are able to undergo the relevant morphogenic program. Therefore, while Zn²⁺ contamination is a more obvious problem when one desires to achieve Zn²⁺ limitation, Zn²⁺ depletion could also present a problem due to the growth impairment and missed Zn²⁺-dependent trigger during active growth. In addition, using monochromator or filter-based plate readers with top- or bottom-reading modes for fluorescence intensity slightly affects the F_375/420 percentage calculated. Therefore, values from each laboratory may not be directly comparable to those presented here, but they should be similar and follow the same trends. Thus, methods and procedures to make ZLM that yields Zn²⁺-limiting growth should be optimized for each laboratory and ultrapure water system, and the bioindicator phenotypes presented here should be used as the standard to define Zn²⁺-limiting conditions.

Due to the difficulty in standardizing and reproducing Zn²⁺-limited growth conditions, an accessible approach to circumvent Zn²⁺ contamination is to add a chelating agent to media prepared without added Zn²⁺. However, this approach confounds the true biological response to Zn²⁺ limitation, often creating an environment starved for Zn²⁺ and other micronutrients. Even Zn²⁺-specific chelating agents are shown to upregulate the response to iron starvation, in addition to the Zn²⁺-dependent transcriptional response in M. tuberculosis²³. Moreover, the use of chelating agents requires careful titration and timing to avoid a severely Zn²⁺-starved environment that does not reflect the biological response during the switch from Zn²⁺-replete to Zn²⁺-limited conditions or the endured growth in environments with limited free Zn²⁺ availability. Finally, adding chelating agents may not be compatible with downstream applications (e.g., macrophage or animal infections). Fundamentally different conclusions have been drawn about the form and function of Alt-ribosomes obtained with chelating agents versus gradual Zn²⁺ depletion in actively growing cultures^17,24. Highlighted here are different phenotypes resulting from relatively minor differences in water purity in different batches of media, even when the procedure is done correctly. The quantifiable, reproducible, and biologically relevant system for generating Alt-ribosomes presented here, i.e., in Zn²⁺-limited wild-type cultures without adding chelators, offers a standardized method that will be paramount to unifying contributions across the field towards a better understanding of alternative ribosome biology.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1000 mL Vacuum Filtration Systems, Standard Line	VWR	10040-440
125 mL Erlenmeyer Shaker Flasks	Greiner Bio-One	679501
14 mL Culture Tubes, Plastic, with Dual-Position Caps	VWR	60818-725
250 mL Erlenmeyer Shaker Flasks	Greiner Bio-One	679502
40X-1000X Lab Binocular Biological Compound Microscope	Omax	M82EZ
50 mL Bio-Reaction Tubes, Sterile	VWR	76211-286
50 mL High-Performance Centrifuge Tubes with Flag Caps	VWR	89039-656
Alcojet Low-Foaming Powdered Detergent	Alconox	1404
Ammonium iron (III) citrate, brown	Thermo Scientific	AAA1119930	MW: 265 g/mol, for 1M solution add 1.325 g to 5 mL ultrapure water; store protected from light at 4 C
Bovine serum albumin	Thermo Scientific	J10857-A1
Calcium D-Pantothenate	Thermo Scientific	AC243300050
Catalase	Sigma-Aldrich	C1345-10G
Citric acid anhydrous, crystalline	Fisher Scientific	BP339-500
Dextrose anhydrous	Fisher Scientific	D14-500
Difco Middlebrook 7H9 Broth	BD Diagnostics	271310
DiluPhotometer OD600 for the Determination of Cell Density and Bradford Assays	Implen	OD600-10
GENios FL Microplate Reader	Tecan		Discontinued
Glycerol	Fisher Scientific	G33-4
Infinite M200 PRO Multimode Microplate Reader	Tecan	LGE140213-20BL
L-Asparagine	Thermo Scientific	AAB2147336
L-Leucine	Thermo Scientific	A1231122
Macrofire SP CCD camera	Optronics		For use with Olympus microscope
Magnesium sulfate heptahydrate	VWR	97062-134
Milli-Q Direct 8 Water Purification System	Millipore	C85358
Moticam X3	Motic		Discontinued, replaced by Moticam X5 Plus; for use with compound microscope
Oleic acid	Thermo Scientific	031997-14
Olympus BX51 upright microscope	Olympus		Discontinued, replaced by BX53M
Potassium phosphate, monobasic	Fisher Scientific	BP362-500
Semi-Micro Two-Sided Disposable Plastic Cuvettes	VWR	97000-590
Sodium chloride, crystal	VWR	VW1494-01
Sodium hydroxide	Fisher Scientific	S318-500
Tween 80	MP Biomedicals	103170
Zinc sulfide heptahydrate, crystalline/ACS	MP Biomedicals	219145290