Jan 29, 2025

Public workspaceBacterial Staining and Microscopy V.2

DOI
dx.doi.org/10.17504/protocols.io.q26g7m62kgwz/v2
  • 1Biotechnology Teaching Program (BIT), North Carolina State University
  • BIT-Protocols
    Tech. support phone: +91 95134-135 email: ccgoller@ncsu.edu
Carlos Carlos Goller
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DOI: dx.doi.org/10.17504/protocols.io.q26g7m62kgwz/v2
Protocol Citation: Carlos Carlos Goller 2025. Bacterial Staining and Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7m62kgwz/v2Version created by Carlos Carlos Goller
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 27, 2025
Last Modified: January 29, 2025
Protocol Integer ID: 119250
Keywords: microscopy, bacteria, gram stain
Funders Acknowledgements:
Biotechnology Program
Abstract
Overview and Goals
Now that you have streaked your unknown bacteria for isolation, you will take steps to identify certain properties of the organisms via a variation of the gram stain. Broadly, bacteria can be categorized into gram-positive and gram-negative groups based on the presence or absence of an outer cell membrane and a thick peptidoglycan layer (cell wall).

Learning Objectives After completing this lab, you will gain the following lab skills:
  • Lab safety and proper personal protective equipment (PPE)
  • Use of gram staining/ gram staining alternative to identify the gram status of a bacterial isolate
  • Use of a fluorescent microscope for the detection of red/green fluorescence

Before starting
Review the complete protocol before beginning. Several of the steps in this procedure are time and/or temperature-sensitive, so you must know what to expect and how to manage your time. Work as a team! There are also several types of tubes with specific names that need to be used for specific protocol steps. They are shown in the illustration below to help you keep track of these materials.
Image Attribution
Image purchased from The Noun Project and recolored by Carlos C. Goller
Guidelines
Clean-Up Instructions
  • All liquids containing bacteria must be poured into a beaker for bleach decontamination (can be found on the blue bench paper by this sink labeled "liquid").
  • All bacterial growth plates can be thrown away in the biohazard trash
  • Return all reusable materials to our storage drawers.
  • Wipe down all surfaces with Conflikt and then ethanol.
  • Remove PPE.
  • Wash hands.
Materials
Each group will need:
Safety warnings
Ask your instructor for instructions on how to disposeof the unused dye solutions.
Before start
Review the complete protocol before beginning. Several of the steps in this procedure are time and/or temperature-sensitive, so you must know what to expect and how to manage your time. Work as a team! There are also several types of tubes with specific names that need to be used for specific protocol steps. They are shown in the illustration below to help you keep track of these materials.

Protocol 1: Invitrogen BacLight Kit
Protocol 1: Invitrogen BacLight Kit
53m
53m
Take bacteria from a plate using a sterile swab.
1m
Combine equal volumes of Component A and Component B in a microfuge tube and mix thoroughly.
2m
Add Amount3 µL of the dye mixture per mL of bacterial suspension.
3m
Mix thoroughly and incubate at room temperature in the dark for 15 minutes. Duration00:15:00 in the dark at room temperature

15m
Incubation
Mix
Trap Amount5 µL of each stained bacterial suspension between a slide and an 18 mm square coverslip.

2m
Observe in a fluorescence microscope equipped with any of the filter sets listed in
Table 1. Live gram-negative organisms should fluoresce green and gram-positive bacteria should fluoresce red.

ABC
-SYTO 9Hexidium iodide
Excitation/Emission480/500 nm518/600 nm
Standard filter setGFPRFP
Storage conditions-20°C, protect from light-20°C, protect from light
Table 1. Live gram-negative organisms should fluoresce green and gram-positive bacteria should fluoresce red. Source: Invitrogen LIVE BacLight kit.

15m
Analyze
Imaging
Cleanup Instructions. All liquids containing bacteria must be poured into a beaker for bleach decontamination (can be found on the blue bench paper by this sink labeled "liquid").
5m
Critical
All bacterial growth plates can be thrown away in the biohazard trash
1m
Return all reusable materials to our storage drawers.
1m
Wipe down all surfaces with Conflikt and then ethanol.
1m
Remove PPE.
1m
Wash hands.


1m
Save images and analyze results.
Note
Critical Thinking Questions: Microscopy Protocol 1
  1. What advantages and disadvantages can you think of when using fluorescent dyes instead of crystal violet (the traditional gram stain dye)?
  2. Why do you think it is important that SYTO9 and Hexidium Iodide not have significantly overlapping emission spectra?
  3. An iodine and then an alcohol wash are the final steps of a gram stain. Based on your understanding of lipid membranes and cell walls, what do you think the purpose of these reagents is? (Hint: iodine creates a high molecular weight complex with crystal violet)
  4. Why might it be important to use a gram stain or acceptable alternative to identify the gram status of a bacterial isolate in a medical setting?

5m
Protocol 2: MycoLight Kit
Protocol 2: MycoLight Kit
1h
1h

Safety information
Review the protocol. Please review the complete protocol before beginning. Several of the steps in this procedure are time—and/or temperature-sensitive, so it’s important that you know what to expect and how to manage your time.

We will thaw all the kit components at room temperature before use and spin down briefly. We have created single-use aliquots to avoid freeze-thaw cycles and stored them at -20°C after preparation. We prepared the MycoLight‱ Red stock solution previously by adding 100 µL of ddH2O into one vial of MycoLight‱ Red (Component B) and mixing well.

Instructors have also prepared the MycoLight‱ dye working solution by mixing equal volume of MycoLight‱ Green (Component A) and MycoLight‱ Red stock solution in a tube.

5m
Grow isolates overnight by picking a colony from an agar plate and inoculating Amount2.5 mL of Tryptic Soy Broth (TSB). Grow at Temperature30 °C for 16-18 hours. Dilute in fresh medium and grow for 2-3 hours. The instructors will perform this step.

Incubation
Overnight
Temperature
We will prepare bacteria samples with concentrations around 107 cells/ml. Grow bacteria into late log phase in an appropriate medium. Note: According to the manufacturer, for E. coli culture, OD600 = 1.0 equals 8 x 108 cells/ml. The instructors will help with this step.
Incubation
Overnight
Remove medium by centrifugation at Centrifigation10.000 x g, 00:10:00 and re-suspend the pellet in ddH2O, adjust bacteria concentration to ~ 107 cells/ml.

10m
Add Amount2 µL MycoLight™ dye working solution to Amount100 µL of the bacterial suspension.

2m
Mix well and incubate in the dark forDuration00:15:00 at room temperature. You can store the solution in a 1.5 ml tube in your drawer.

15m
Remove the working solution by centrifugation at Centrifigation10000 x g, 00:10:00

10m
Resuspend the bacteria pellet in Amount100 µL volume of ddH2O.

1m
AddAmount10 µL of stained cells to a clean slide and cover with a coverslip. Provide the rest of the stained cells to your instructor for imaging. Table 2. MycoLight Bacterial Staining

ABC
MycoLight GreenMycoLight Red
Excitation/Emission488/530 nm650/669 nm
Standard filter setFITCCy5
Storage conditions-20°C, protect from light-20°C, protect from light
StainingGram-negative. Example: Escherichia coliGram positive. Example: Bacillus subtilis cells
Table 2. MycoLight Bacterial Staining

2m
Pipetting
Monitor the fluorescence of bacteria with a fluorescent microscope or the Agilent LionHeart.
Note
Critical Thinking Questions: Microscopy Protocol 2
  1. What do your results suggest about your isolate?
  2. What shape does your organism have?
  3. Do your results support the previous staining procedure we used? Why or why not?

15m
Analyze
Imaging