8.1: Introduction

The Birth of Bacteriology

While perhaps best known to us as a cause of human disease, bacteria really should be far more famous for their positive contributions than for their negative ones.

Bacteria were first observed by Anton von Leeuwenhoek in the late 17th century, but didn’t become the objects of serious scientific study until the 19th century, when it became apparent that some species caused human diseases. The methods devised by Robert Koch, Louis Pasteur, and their associates during the “Golden Age” of microbiology, which spanned from the mid-1800s to early 1900s, are still widely used today. Most of these methods involved isolating single bacteria derived from a natural source (such as a diseased animal or human) and cultivating them in an artificial environment as a pure culture to facilitate additional studies.

During the middle of the twentieth century, when we believed we had defeated them at their disease-causing game, bacteria became popular subjects of empirical study in fields such as genetics, genetic engineering, and biochemistry. With the evolution of antibiotic-resistant strains and our increased knowledge of bacterial stealth attack strategies such as biofilms and intracellular growth, medical researchers have refocused their attention on disease-causing bacteria and are looking for new ways to defeat them.

Growing bacteria in pure culture is still one of the most widely used methods in microbiology. Many bacteria, particularly those that cause diseases and those used in scientific studies, are heterotrophic, which means that they rely on organic compounds as food to provide energy and carbon. Some bacteria also require added nutritional components such as vitamins in their diet. An appropriate physical environment must be created, where important factors such as temperature, pH, and the concentration of atmospheric gases (particularly oxygen) are controlled and maintained.

The nutritional needs of bacteria can be met through specialized microbiological media that typically contain extracts of proteins (as a source of carbon and nitrogen), inorganic salts such as potassium phosphate or sodium sulfate, and in some cases, carbohydrates such as glucose or lactose. For fastidious bacteria (meaning, those that are picky eaters) vitamins and/or other growth factors must be added as well.

Figure \(\PageIndex{1}\): Different liquid and solid culture media types. Image credit: Microbiology: A Laboratory Experience by Holly Ahern, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

Bacteriological culture media can be prepared as a liquid (broth), a solid (plate media or slant media), or as a semi-solid (deeps) as illustrated in the figure above. Solid and semi-solid media contain a solidifying agent such as agar or gelatin. Agar, which is a polysaccharide derived from red seaweed (Rhodophyceae) is preferred because it is an inert, non-nutritive substance. The agar provides a solid growth surface for the bacteria, upon which bacteria reproduce until the distinctive lumps of cells that we call colonies form.

Koch, Pasteur, and their colleagues in the 19th and early 20th centuries created media formulations that contained cow brains, potatoes, hay, and all sorts of other enticing microbial edibles. Today, bacteriological media formulations can be purchased in powdered form, so that all the preparer has to do is to measure out the correct amount, add the right amount of water, and mix. After the basic formula has been prepared, the medium is sterilized in an autoclave, which produces steam under pressure and achieves temperatures above boiling. Once sterilized media has cooled, it is ready to be used.

Growing Bacteria in Culture

A population of bacteria grown in the laboratory is referred to as a culture. A pure culture contains only one single type; a mixed culture contains two or more different bacteria. If a bacterial culture is left in the same media for too long, the cells use up the available nutrients, excrete toxic metabolites, and eventually the entire population will die. Thus bacterial cultures must be periodically transferred, or subcultured, to new media to keep the bacterial population growing.

Microbiologists use subculturing techniques to grow and maintain bacterial cultures, to examine cultures for purity or morphology, or to determine the number of viable organisms. In clinical laboratories, subculturing is used to obtain a pure culture of an infectious agent, and also for studies leading to the identification of the pathogen. Because bacteria can live almost anywhere, subculturing steps must be performed aseptically, to ensure that unwanted bacterial or fungal contamination is kept out of an important culture.

In microbiology, aseptic techniques essentially require only common sense and good laboratory skills. First, consider that every surface you touch and the air that you breathe may be contaminated by microorganisms. Then think about the steps you can take to minimize your exposure to unwanted invisible intruders. You should also be thinking about how to prevent contamination of your bacterial cultures with bacteria from the surrounding environment (which includes you).

To maintain an aseptic work environment, everything you work with should be initially free of microbes. Thus, we begin with pre-sterilized pipettes, culture tubes, and glassware. Inoculating loops and needles made of metal wire can be used to transfer bacteria from one medium to another, such as from the surface of an agar plate to a broth. Metal tools may be sterilized by heating them in the flame of a Bunsen burner. Glass tools or metal spreaders or forceps that can’t be sterilized by direct heat are dipped in alcohol followed by a brief pass through the flame to speed the evaporation process. Standard aseptic techniques used for culturing bacteria will be demonstrated at the beginning of lab.

Figure \(\PageIndex{2}\): Colonies of bacteria growing on an agar plate. Image credit: Microbiology: A Laboratory Experience by Holly Ahern, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

One very important method in microbiology is to isolate a single type of bacteria from a source that contains many. The most effective way to do this is the streak plate method, which dilutes the individual cells by spreading them over the surface of an agar plate (see Figure 2). Single cells reproduce and create millions of clones, which all pile up on top of the original cell. The piles of bacterial cells observed after an incubation period are called colonies. Each colony represents the descendants of a single bacterial cell, and therefore, all of the cells in the colonies are clones. Therefore, when you transfer a single colony from the streak plate to new media, you have achieved a pure culture with only one type of bacteria.

Different bacteria give rise to colonies that may be quite distinct to the bacterial species that created it. Therefore, a useful preliminary step in identifying bacteria is to examine a characteristic called colonial morphology, which is defined as the appearance of the colonies on an agar plate or slant. Ideally, these determinations should be made by looking at a single colony; however, if the colonial growth is more abundant and single colonies are absent, it is still possible to describe some of the colonial characteristics, such as the texture and color of the bacterial growth.

Describing Colonial Morphology of Bacteria

By looking closely at the colonial growth on the surface of a solid medium (such as a petri plate), characteristics such as surface texture, transparency, and the color or hue of the growth can be described. The following three characteristics are readily apparent whether you’re looking at a single bacterial colony or more dense growth, without the aid of any type of magnifying device.

Texture—describes how the surface of the colony appears. Common terms used to describe texture may include smooth, glistening, mucoid, slimy, dry, powdery, flaky etc.

Transparency—colonies may be transparent (you can see through them), translucent (light passes through them), or opaque (solid-appearing).

Color or Pigmentation—many bacteria produce intracellular pigments which cause their colonies to appear a distinct color, such as yellow, pink, purple or red. Many bacteria do not produce any pigment and appear white or gray.

Figure \(\PageIndex{3}\): Bacteriological descriptions of colonial morphology. Image credit: Microbiology: A Laboratory Experience by Holly Ahern, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

As the bacterial population increases in number, the colonies get larger and begin to take on a shape or form. These can be quite distinctive and provide a good way to tell colonies apart when they are similar in color or texture. The following three characteristics can be described for bacteria when a single, separate colony can be observed. It may be helpful to use a magnifying tool, such as a colony counter or dissecting microscope, to enable a close-up view of the colonies. Colonies should be described as to their overall size, their shape or form, what a close-up of the edges of the colony looks like (edge or margin of the colony), and how the colony appears when you observe it from the side (elevation).

Figure 4 shows a close-up of colonies growing on the surface of an agar plate. In this example, the differences between the two bacteria are obvious, because each has a distinctive colonial morphology.

Figure \(\PageIndex{4}\): Two different organisms growing colonies on an agar surface. Image credit: Microbiology: A Laboratory Experience by Holly Ahern, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

All of the above content was adapted from https://milnepublishing.geneseo.edu/suny-microbiology-lab/chapter/bacteriological-culture-methods/

Viewing Bacterial Cells

The microscope is a very important tool in microbiology, but there are limitations when it comes to using one to observe cells in general and bacterial cells in particular. Two of the most important concerns are resolution and contrast. Resolution is a limitation that we can’t do much about, since most bacterial cells are already near the resolution limit of most light microscopes. Contrast, however, can be improved by either using a different type of optical system, such as phase contrast or a differential interference contrast microscope, or by staining the cells (or the background) with a chromogenic dye that not only adds contrast, but gives them a color as well.

There are many different stains and staining procedures used in microbiology. Some involve a single stain and just a few steps, while others use multiple stains and a more complicated procedure. Before you can begin the staining procedure, the cells have to be mounted (smeared) and fixed onto a glass slide.

A bacterial smear is simply that—a small amount of culture spread in a very thin film on the surface of the slide. To prevent the bacteria from washing away during the staining steps, the smear may be chemically or physically “fixed” to the surface of the slide. Heat-fixing is an easy and efficient method, and is accomplished by passing the slide briefly through the flame of a Bunsen burner, which causes the biological material to become more or less permanently affixed to the glass surface.

Heat fixed smears are ready for staining. In a simple stain, dyes that are either attracted by charge (a cationic dye such as methylene blue or crystal violet) or repelled by charge (an anionic dye such as eosin or India ink) are added to the smear. Cationic dyes bind the bacterial cells which can be easily observed against the bright background. Anionic dyes are repelled by the cells, and therefore the cells are bright against the stained background. See Figures 6 and 7 for examples of both.

Figure \(\PageIndex{5}\): Negative stain of Cyptococcus neoformans, an encapsulated yeast. Image credit: Microbiology: A Laboratory Experience by Holly Ahern, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

Figure \(\PageIndex{6}\): Positive stain of Staphylococcus aureus. Image credit: Microbiology: A Laboratory Experience by Holly Ahern, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

Probably the most important feature made obvious when you stain bacterial cells is their cellular morphology (not to be confused with colonial morphology, which is the appearance of bacterial colonies on an agar plate). Most heterotrophic and culturable bacteria come in a few basic shapes: spherical cells (coccus/cocci), rod-shaped cells (bacillus/bacilli), or rod-shaped cells with bends or twists (vibrios and spirilla, respectively). There is greater diversity of shapes among Archaea and other bacteria found in ecosystems other than the human body.

Often bacteria create specific arrangements of cells, which form as a result of binary fission by the bacteria as they reproduce. Arrangements are particularly obvious with non-motile bacteria, because the cells tend to stay together after the fission process is complete. Both the shape and arrangement of cells are characteristics that can be used to distinguish among bacteria. The most commonly encountered bacterial shapes (cocci and bacilli) and their possible arrangements are shown in the Figure below.

Figure \(\PageIndex{7}\): Bacterial Shapes and Arrangements. Image by McLaughlin and Petersen, work is made publicly available by the City University of New York (CUNY). Contact: [e-mail].

https://academicworks.cuny.edu/cgi/viewcontent.cgi?referer=&httpsredir=1&article=1015&context=qb_oers

Differential Staining Techniques

In microbiology, differential staining techniques are used more often than simple stains as a means of gathering information about bacteria. Differential staining methods, which typically require more than one stain and several steps, are referred to as such because they permit the differentiation of cell types or cell structures. The most important of these is the Gram stain. Other differential staining methods include the endospore stain (to identify endospore-forming bacteria), the acid-fast stain (to discriminate Mycobacterium species from other bacteria), a metachromatic stain to identify phosphate storage granules, and the capsule stain (to identify encapsulated bacteria). We will be performing the Gram stain and endospore staining procedures in lab, and view prepared slides that highlight some of the other cellular structures present in some bacteria.

Gram Stain

Figure \(\PageIndex{8}\): Gram positive (purple) and Gram negative (pink) stained cells. Image credit: Microbiology: A Laboratory Experience by Holly Ahern, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

In 1884, physician Hans Christian Gram was studying the etiology (cause) of respiratory diseases such as pneumonia. He developed a staining procedure that allowed him to identify a bacterium in lung tissue taken from deceased patients as the etiologic agent of a fatal type of pneumonia. Although it did little in the way of treatment for the disease, the Gram stain method made it much easier to diagnose the cause of a person’s death at autopsy. Today we use Gram’s staining techniques to aid in the identification of bacteria, beginning with a preliminary classification into one of two groups: Gram positive or Gram negative.

The differential nature of the Gram stain is based on the ability of some bacterial cells to retain a primary stain (crystal violet) by resisting a decolorization process. Gram staining involves four steps. First cells are stained with crystal violet, followed by the addition of a setting agent for the stain (iodine). Then alcohol is applied, which selectively removes the stain from only the Gram-negative cells. Finally, a secondary stain, safranin, is added, which counterstains the decolorized cells pink.

Although Gram didn’t know it at the time, the main difference between these two types of bacterial cells is their cell walls. Gram negative cell walls have an outer membrane (also called the envelope) that dissolves during the alcohol wash. This permits the crystal violet dye to escape. Only the decolorized cells take up the pink dye safranin, which explains the difference in color between the two types of cells. At the conclusion of the Gram stain procedure, Gram positive cells appear purple, and Gram-negative cells appear pink.

When you interpret a Gram-stained smear, you should also describe the morphology (shape) of the cells, and their arrangement. In Figure 8.1.8, there are two distinct types of bacteria, distinguishable by Gram stain reaction, and also by their shape and arrangement. Can you describe them?