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Kevin Hughes Author
Kevin Hughes is a research engineer with 10 years of industrial R&D experience, in the fields of semiconductor manufacturing, complex fluids, and materials science. He has a BS from Carnegie Mellon University and a PhD from Cornell University, both in Chemical Engineering. His academic research involved controlling the formation of interfaces between materials on a molecular scale using various types of engineered surface chemistry, including self-assembled monolayers. In industry, he continued to work on a variety of related problems, mostly involving the control of chemical and physical processes on the micro and nano scale for applications in manufacturing and product development. He has authored several academic and technical publications, in the fields of thin film materials science and physical chemistry. He is passionate about teaching, and making science and technology concepts accessible to everyone.
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Kevin Hughes Author
Kevin Hughes is a research engineer with 10 years of industrial R&D experience, in the fields of semiconductor manufacturing, complex fluids, and materials science. He has a BS from Carnegie Mellon University and a PhD from Cornell University, both in Chemical Engineering. His academic research involved controlling the formation of interfaces between materials on a molecular scale using various types of engineered surface chemistry, including self-assembled monolayers. In industry, he continued to work on a variety of related problems, mostly involving the control of chemical and physical processes on the micro and nano scale for applications in manufacturing and product development. He has authored several academic and technical publications, in the fields of thin film materials science and physical chemistry. He is passionate about teaching, and making science and technology concepts accessible to everyone.

Introduction

In most types of microscopy, the most complicated and sensitive aspect of the analysis is the preparation of specimens. The visualization of microorganisms using optical microscopy is no exception. Microorganisms are generally transparent, fragile, and highly sensitive to their environments, and for that reason, careful preparation is required to visualize them without significantly perturbing their structure.

In this article, we will discuss some of the most important and useful specimen preparation techniques in modern optical microscopy. First, we will cover the methods used for immobilizing and selectively staining cells or parts of cells. In the second part, we will describe some methods that can be used to visualize living cells, and some practical aspects of using an optical microscope to carry out analysis.

Staining

Since microorganisms are mostly transparent, staining can be very helpful in visualizing them, including their internal structures. Staining involves adding a single dye, or sequence of dyes, which selectively attach to parts of the cell either chemically (like an acid-base reaction), or through physical processes (by being trapped inside the cell, for example). Some dyes also improve contrast by changing the nature of the background field away from the cell.

In addition to improving visualization of cell structures, staining can also be used as a tool to identify unknown organisms based on their reactions to staining procedures. The Gram stain, probably the most important type of staining known in microbiology, is an example of this and will be described in detail below.

In this section, we will first describe the preparation of a smear, which is typically used to generate specimens for staining, then discuss some of the more commonly used staining techniques.

Smear preparation

For most staining procedures, the first step involves the preparation of a specimen by making a smear. To summarize, smearing is a simple technique that involves the placement of a small sample of the microorganism on a slide, dispersing it, and then drying and heating it to fix or immobilize the cells onto the slide surface. The procedure also kills the cells. Dispersing and drying the cells enhances the uptake of the dye used in staining, while heat fixing is required to prevent cells from being washed off the slide during staining and subsequent rinsing.

Care is required to generate a specimen of optimal thickness, and to not contaminate the specimen or any lab equipment that may be used for subsequent analysis.

There are a number of possible variations on the procedure used for smear preparation, but in general, the following steps can be used [1][2]:

  1. Gather materials, and prepare the work area and any slides you plan to use. Clear off an area of the lab bench and lay down a paper towel in the work area.
  2. Prepare any slides you plan to use. Clean the slides using soap and water, and thoroughly rinse and dry them, taking care to either blow dry or wipe with a lint-free cloth to avoid forming water spots.
  3. Draw circles using a wax pencil on the slides. These circles will define the target areas on the slide for the specimens, and also help to confine the specimens to these areas. It is also helpful to mark the slide with any description of the samples (for example, lab notebook page numbers, the date, or your initials).
  4. Pre-clean an inoculation loop using a bunsen burner flame, and allow it to cool. (A sterile disposable tool can be used in place of an inoculation loop, for example, a swab.)
  5. If the samples are from a solid medium (an agar culture, for example), place 1-2 drops of water in the wax circle using the inoculation loop. For broth (liquid) cultures, this step is not needed.
  6. Gather a small portion of the sample onto the loop, and place it onto the slide in the wax circle area. If the sample is solid, gently mix it into the water drop using the transfer tool. If the sample is coming from a liquid broth, it is advisable to mix the broth using a vortex mixer or other device prior to sampling.
  7. Allow the sample to air dry. This will prevent splattering of the specimen in the next step.
  8. Heat the slide by passing it over a bunsen burner briefly 3-4 times, specimen side up, or by placing it into a microincinerator.
  9. If a loop was used, re-sterilize it by holding it in the bunsen burner flame until glowing red.

A picture of a finished bacterial smear is shown in Figure 1.

Figure 1: Three bacterial smear samples, reproduced from.[2]

If multiple specimens are being prepared on a single slide, deposit the samples and air dry all of them prior to flaming. Trial and error is sometimes needed to obtain the optimal thickness or concentration of cells in the smear. This can be controlled by diluting with water or saline on the slide, as described in steps 5 and 6, or by obtaining more of less of the sample on the transfer tool in step 6.

Types of Stains

There are a number of staining techniques which have been developed over many years for the accurate imaging of specific organisms and cellular structures. Here, we will provide descriptions of the most common and useful. References 1 and 3 provide excellent and exhaustive lists of available stains.

Simple Stain

This is a technique that only uses a single stain, and is useful for simply visualizing cells, to determine characteristics like size, shape, and count. In this method, the stain has a basic functionality, causing it to become positively charged in a neutral pH medium. Most cells acquire a negative charge under similar conditions, so the stain selectively attaches to the cell surface.

Examples of stains that can be used for this technique include crystal violet, safranin, and methylene blue.

Negative Stain

A negative stain works in the opposite way. These stains are acidic, and therefore become negatively charged at a neutral pH. Instead of staining the cells, they are repelled from the cells and attach and stain the rest of the specimen. When using a negative stain, cells appear bright on a dark background.

Nigrosin is a stain that can be used in this way.

Gram Stain

Gram staining is a more complex, multi-step technique, but is a powerful method for classifying bacteria.[3] The technique involves exposing the cells to a primary stain, crystal violet, then exposing them to an iodine solution, which forms a complex with the stain. All cells will take up crystal violet and form this complex. Next, the specimen is exposed to a decolorizer, which is either ethanol, or a mixture of acetone and ethanol. In some cells, called ‘gram negative’ cells, the decolorizer will dissolve the stain-iodine complex and carry it out of the cell. In ‘gram positive’ cells, the complex cannot pass back through the cell wall, and is trapped inside the cell. Finally, a second stain is added to re-stain the gram negative cells, typically safranin or carbol fuchsin. The second stain will also enter gram-positive cells, but it is lighter in color so the staining in those cells is dominated by the darker purple crystal violet-iodine complex. A schematic summary of the Gram staining procedure, with an example of a stained specimen, is shown in Figure 2.

The staining procedure therefore classifies cells based on the reaction of the cell walls to the decolorizing solvent. Gram positive cells, which show up purple, have a high concentration of sugar-protein complexes in their cell walls, while gram negative cells, which show up pink or red, have a low concentration of these complexes.

The Gram staining procedure can often lead to equivocal results. For example, if the smear is too thick, this can result in incomplete decolorization due to transport limitations of the decolorizing agent. In other cases, if the culture is too old, results can be variable. Therefore, the most complete guidelines for the staining procedure usually include advice to:

  • Use relatively young cultures
  • Prepare light, thin films

If possible, repeat the same smearing and Gram staining procedure on two additional control samples- one that is known to be Gram-negative, the other known to be Gram-positive.

Figure 2: top, a schematic of the Gram staining procedure; [1] bottom, example of a specimen stained using the Gram procedure.[4]

Acid-fast staining

Acid-fast staining is a similar method to Gram staining, but adapted for organisms with significantly nonpolar and impenetrable cell walls.[1] Examples of such organisms are members of the genus Mycobacterium. The thickness and waxy nature of the cell walls of these organisms make it difficult to stain using the standard Gram technique.

In this procedure, the specimen is first exposed to carbol fuchsin and heated (or alternatively, exposed to a lipid solubilizer). This stains the cell. In the second step, the specimen is exposed to an acid-alcohol solution. Only cells with a sufficiently impenetrable cell wall will retain the dye in this step, and these cells are said to be acid fast. A second counter-stain can then be used to re-stain the decolorized organisms, similar to the Gram staining procedure.

Flagella stain

Some microorganisms have one or more small, thin appendages that are used to move the organism around in a liquid. These appendages, called flagella, are far below the resolution limit for optical microscopy, at typically < 30 nm in thickness. Therefore, special staining procedures are necessary to visualize these structures using optical microscopy. One such procedure uses a simple basic stain dissolved in ethyl alcohol.[5] As with other stains for flagella, the technique requires rigorously cleaned microscope slides and otherwise very careful, detail-oriented technique.

An alternative method for visualizing such small structures is the use of electron microscopy.

Lab Procedures for Staining

As discussed above, there are many staining procedures that can be used, all with their own chemistry and step-by-step procedure. As an example of a common procedure, the following steps can be used for Gram staining.[6]

  1. Make a smear specimen as described in the previous section. When preparing an unknown specimen, prepare a known gram-positive and gram-negative specimen along side the unknown sample and carry through the staining procedures on those smears also.
  2. Place a few drops of crystal violet stain onto the smear, to completely cover it. Let the slide sit for or 1 minute, then rinse with water. The rinsing can be done by dipping the slide into a beaker of water (or multiple beakers if needed).
  3. Repeat step #2, using Gram’s iodine solution in place of crystal violet.
  4. Rinse the smear with the decolorizer (ethanol or an acetone-ethanol mixture) for roughly 5 seconds, then rinse with water. Be very careful in this step to not overexpose the smear by rinsing with decolorizer for too long. This can have the effect of removing stain from the gram-positive organisms.
    This can be accomplished by holding the slide at an angle, and letting the decolorizer flow over the stain until the decolorizer flowing off the slide is clear. At this point, quickly dip the slide into water.
  5. Expose the smear to safranin or fuchsin for 1 minute, then rinse with water.
  6. Allow the slide to dry completely in air, or blot it dry.

Other Specialized Techniques for Analyzing Micro-organisms with Optical Microscopy

The hanging drop technique

A disadvantage to using the smear / stain technique described above is that it kills and immobilizes the microorganisms, which prevents analysis of motility and other related properties. By using so called “wet mount” techniques, the microorganisms can be kept in a liquid environment in which they are alive during analysis.

The simplest type of wet mount method is to place a drop of liquid containing the microorganism onto a slide, then gently placing a coverslip over the drop. This method is susceptible to drying, however. A more complex but stabler type of wet mount is the hanging drop.[7] In this method, a liquid droplet with the microorganism is hanging down below the coverslip, with the sample sealed from atmosphere using a ring of wax or petroleum jelly. An illustration of a hanging drop mount is shown in Figure 3.

Figure 3: hanging drop mount (side view)[7]

A hanging drop mount can be made using the following procedure:

  1. Holding it by its edges, apply a small amount of petroleum jelly to the corners of a cover slip.
  2. Using an inoculation loop, transfer a small amount of a liquid sample in the middle of the coverslip.
  3. Place a well side, with the concave side down, over the coverslip, so that the sample droplet is centered in the well and the petroleum jelly forms a seal between the slide and cover slip.
  4. Flip the assembly over so the coverslip is on top.

Analysis of hanging drop samples will sometimes involve judgements of cell motility, or the ability of the cells to self-propel. Even non-motile or dead cells can move in liquid, due to convective flow of the liquid, or by Brownian motion. So any analysis of cell motility should be done by carefully observing cell movement over time, to ensure any movement is true motility.

Hemocytometers

A hemocytometer is a special type of microscope slide that can be used for quantitative counting and sizing of cells. It is commonly used to determine the concentration of blood (thus the ‘hemo-’ prefix), sperm, or yeast, but can be used on any type of cell or other particle that can be seen in an optical microscope.

The principle of the hemocytometer is that it consists of chambers that are marked into regions of known volume. The counting chamber, and an example of the marked regions, are shown in Figure 4. By counting the numbers of cells within one or more known volumes, the concentration of cells can be determined (cells per unit volume). A schematic view from the side and top of a hemocytometer is shown in Figure 4.

The device itself, also shown in Figure 4, is a thick microscope slide, with an indented well in the center that serves as the counting chamber, and deeper depressions to capture any overflow of sample liquid. Besides the special slide, a coverslip that is thicker than the standard must be used, so that the surface tension of the liquid under analysis does not deform it.

Figure 4: (a) schematic of the side view of a hemocytometer,[8] (b) photo of a commercial hemocytometer slide,[9] and (c) example layout of the etched lines in the counting chamber.[9]

Determining cell concentration using a hemocytometer involves first thoroughly agitating the sample to ensure it is fully mixed, then making the proper dilution. Because the technique relies on visually counting cells, overlapping or agglomerated cells can artificially reduce the measured concentration. So a high dilution must be used if the original sample is very concentrated.

After diluting and placing the sample into the hemocytometer, the next step is to count the cells in a given set of demarcated regions. Enough cells should be counted in order to obtain a statistically significant sample size, which for most analyses is on the order of ~ hundreds of cells. The analyst must also be systematic in whether or not to count cells that lie on a demarcation line. For example, one system is to count cells that line only on the lower or left side of a given area.

Ocular and Stage Micrometers

An ocular micrometer is an optical element that is placed in the eyepiece of a microscope, which superimposes marks of known pitch onto the magnified image (the pitch is the distance between marks). Ocular micrometers are a subset of a type of optical element known as a reticle; examples of other types of reticles are shown in Figure 5.

Figure 5: various types of reticles for optical microscopes, including ocular micrometers (2nd and 4th reticles) [10]

By knowing the pitch and the total magnification, the analyst can estimate distances in the field of view, and use that to estimate the sizes or other dimensions of microorganisms, or any other specimen under analysis. For example, if an optical micrometer has a pitch of 100 um, and 10X magnification is being used, then the distance between marks on the magnified image is 10 μm (100 um / 10).

However, this approach is often imprecise since the exact magnification and pitch are not known. For this reason, a stage micrometer should be used to calibrate the ocular micrometer. This is a special microscope slide with a printed scale showing absolute distances. The ocular micrometer can be calibrated using this tool by comparing the distance between marks on the ocular micrometer, and marks on the stage micrometer, using various objectives.

Operation of the Optical Microscope

In this section, we will describe the steps for basic operation of an optical microscope. These steps can be thought of as general for any of the specimen preparation techniques described here.

  1. Develop a plan and set of goals for the analysis, or simply write the questions you hope to answer.
  2. Clean the microscope slide and cover slip. This is especially important for analyzing microorganisms, where contamination can significantly affect results.
  3. Generate a specimen using one of the techniques discussed above. Common to all of the techniques are the following:
    1. Make the specimen from a representative sample of the microorganism. For liquid samples, this involves thoroughly mixing.
    2. Use an appropriate dilution, to avoid overlap or agglomeration of cells. This may require trial and error.
  4. Once the specimen is prepared, begin by observing the sample at low magnification using dry objectives. For unstained samples (those made with a wet mount, for example), using very low illumination will help to visualize partially transparent organisms.
  5. Progressively increase the magnification, being careful not to crash the objectives into the slide. Higher magnifying power objectives come closer to the sample, so there is more risk that they can contact the coverslip.
  6. If needed, apply immersion oil and switch to an immersion objective.
  7. If a digital camera is installed on the microscope, collect representative images during the analysis.
  8. After completing the analysis, either store the slide safely or dispose it according to the procedures of your lab.

References

  1. Pommerville, J. C. (2010). Alcamo’s laboratory fundamentals of microbiology. Jones & Bartlett Publishers.
  2. Association of Public Health Laboratories, AFB Smear Microscopy Trainer Notes: https://www.aphl.org/programs/infectious_disease/tuberculosis/TBCore/TB_AFB_Smear_Microscopy_TrainerNotes.pdf
  3. Reddy, C. A., Beveridge, T. J., Breznak, J. A., & Marzluf, G. (Eds.). (2007). Methods for general and molecular microbiology. American Society for Microbiology Press.
  4. American Society for Microbiology, “Examination of Gram Stains of Bacterial Skin Infections”,  http://www.asmscience.org/content/education/imagegallery/image.3064
  5. Clark, W. A. (1976). A simplified Leifson flagella stain. Journal of clinical microbiology, 3(6), 632.
  6. Bio-Rad Laboratories, “Gram Staining”, https://www.youtube.com/watch?v=sxa46xKfIOY&t=190s
  7. Microbeonline Medical Microbiology Guide, “Procedure of Hanging Drop method to test Bacterial Motility”, https://microbeonline.com/procedure-hanging-drop-method-test-bacterial-motility/
  8. Microbehunter Microscopy, “The hemocytometer (counting chamber)”, http://www.microbehunter.com/the-hemocytometer-counting-chamber/
  9. Weber Scientific, “Hemocytometer for Cell Counting”, https://www.weberscientific.com/hemocytometer-for-cell-counting
  10. Edmund Optics, “Reticles and Stage Micrometers”, https://www.edmundoptics.com/c/reticles-stage-micrometers/707/

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