microbiology module 4

What are the three forms of hemolysis found on blood agar plates?

Alpha (green), Beta (clear zones) and Gamma (white/tan). Microbes capable of alpha hemolysis present with greenish-brown color colonies due to the incomplete (partial) lysis of red blood cells. Beta hemolysis is classified as the capacity of a microbe to completely lyse red blood cells. The resulting lysis presents as a distinct zone of clearing around the growing colony. Gamma hemolysis designates the absence or lack of hemolytic activity and the resulting colonies are often white/tan in color growing on the red background color of the unaffected blood agar plate.

What are the requirements of a fastidious microbe?

A fastidious microbe is an organism with complex growth requirements such that if absent it will not grow. Enriched medias thus contain these specific and essential nutrients required for the growth of a particular subset of microorganisms.

Microbial Growth and Control

An important aspect within the field of microbiology is the ability to cultivate microorganisms. Consider the following scenario: You are flying over a field, and you are trying to see someone on the ground holding a red flag. Would it be easier to see a single flag or 10 million flags in the same field? The latter would be much easier to detect. The same principle often applies in microbiology. While many tests are highly sensitive, often times expanding a given microbial population allows researchers to detect and assess the microorganism more easily. In the following module, we will first discuss the various techniques used to promote microbial growth, how to differentiate between samples, and how researchers safely control its growth.

What is the primary purpose of Columbia CNA agar?

As it suppresses the growth of Gram-negative bacteria CNA agar is, therefore, used for isolation of Gram-positive microbes.

Blood Agar

Blood agar, also known as BAP (blood agar plates), is a derivative of TSA whereby mammalian blood, often from sheep, is added to the plate composition at a concentration range of 5-10%. As such, BAP have a distinct red coloration. BAP is an enriched, non-selective yet differential media. The red blood cells in the media promote the growth of fastidious microorganisms, such as various strains of Streptococcus. BAP is a differential media used to detect hemolytic activity. Hemolysis, the lysis (breakdown) of red blood cells, is classified as alpha, beta, or gamma. When grown on BAP, microbes capable of alpha hemolysis present as greenish-brown color colonies due to the incomplete (partial) lysis of red blood cells. Beta hemolysis is classified as the capacity of a microbe to completely lyse red blood cells. The resulting lysis presents as a distinct zone of clearing around the growing colony. Gamma hemolysis designates the absence or lack of hemolytic activity, and the resulting colonies are often white/tan in color growing on the red background color of the unaffected blood agar plate.

Chocolate Agar

Chocolate agar, also known as CHOC or as chocolate blood agar (CBA), is an enriched, non-selective, non-differential derivative of blood agar plates. Unlike blood agar plates that contain intact red blood cells, chocolate agar plates contain red blood cells that have been lysed by heat. This cooking of the red blood cells gives the media a dark brown chocolaty appearance, hence the name. Note: Actual chocolate is never added to the agar. The lysed red blood cells release various growth factors into the media required for the cultivation of fastidious, pathogenic bacteria. As such, chocolate agar is also considered an enriched media. Examples include the Gram-negative bacteria Haemophilus influenzae and Neisseria meningitidis.

Columbia CNA Agar

Columbia CNA agar is red in color and is an enriched, selective, and differential media. As the media contains the antimicrobial agents colistin and Nalidixic acid, it suppresses the growth of Gram-negative bacteria and is, therefore, used for isolation of Gram-positive microbes. Similar to BAP, the media is also enriched with blood, which allows for differentiation based on hemolytic patterns.

Differential Media

Differential media distinguishes between two, often related, microbes. For instance, two microbes Escherichia coli and Salmonella are both Gram-negative but can be distinguished by the presence (or absence) of lactose fermentation. If grown on the same differential media, E. coli ferments lactose and turns the culture red. In contrast, Salmonella does not ferment lactose, and the culture remains white/tan in color. The ability to differentiate within all Gram-negative (or all Gram-positive) species will be further demonstrated in the Lab section of this course.

What is differential media?

Differential media is used to distinguish between two (or more) related microbes.

Enriched Media

Enriched media is used to grow fastidious microorganisms—organisms with complex growth requirements such that if absent will not grow. Thus, enriched medias contain the essential nutrients required for the growth of this subset of microorganisms. Medias can be a combination of selective, differential, and enriched.

Eosin Methylene Blue Agar

Eosin Methylene Blue agar, also known as EMB, is red in color and is classified as both a selective and differential form of media. As the naming suggests, EMB contains eosin and methylene blue that restricts the growth of Gram-positive bacteria. Microorganisms are differentiated based on their ability to ferment the lactose present in the media. Similar to MacConkey agar, non-fermenting (lac-) microbes grow as white/tan colonies, while lactose-fermenting microbes produce dark (purple to black) colonies on the surface of the agar. Most noticeably, if Escherichia coli is grown on EMB agar plates, colonies will have a distinctive metallic green sheen.

True or False: LB agar is classified as a non-selective, differential media.

False

Agar Plates

Growth media comes in two forms: liquid and solid. Solid growth media, often contained within a sterile petri dish, is in its simplest form liquid growth media that has had a hardening agent added. The addition of a polysaccharide derived from seaweed (algae) extract creates a solid medium called agar. Agar is used to create a solid, smooth surface on which microbes can grow. As they grow, microbes form colonies that take on the appearance of individual isolated dots. However, as the microbe increases in number, the isolates merge into one another. Once the microbe has grown to cover the entire plate, this is referred to as a lawn. As we will see below, various forms of agar plates exist in order to aid in the growth and classification of a broad range of microorganisms.

What is another name for a liquid growth media, and what are the basic components?

Growth media in a liquid form is often referred to as nutrient broth. As the name implies its composition consists primarily of essential 'ingredients' such as a rich source of sugars, amino acids and vitamins. Together, a nutrient broth provides the microbe with a source of carbon/energy, which, in turn, encourages its expansion.

Growth Media

Growth media is a liquid or solid formulated to support microbial growth. The media contains essential nutrients to provide the microbe with a source of carbon/energy, which, in turn, encourages its expansion. The nutrient composition often includes a rich source of sugars, amino acids, and vitamins. Such media can also be referred to as a nutrient broth and is commonly used to grow microbes in a suspension. Perhaps the most common nutrient broth in the lab, LB media (lysogeny broth), is a nutritionally rich liquid known for its ability to grow a vast array of microbes. However, there are also occasions where a researcher may want to specifically control what types of microbes can grow. For instance, a researcher may want to establish conditions where various types of bacteria will grow, while others will not (selective). On the other hand, a researcher may want to establish conditions where several types of microbes can be grown simultaneously while being able to distinguish one type from another (differential). Altogether, these conditions would require specialized media. The two general classifications of specialized media (selective vs. differential) are expanded on below.

Scenario #1 A patient is brought into the hospital with severe abdominal pain. Upon examination, the doctor believes the cause of the discomfort to be bacterially related (e.g. food-borne bacterial illness). The doctor then orders bacterial swab samples to be taken from the patient. Once the causative agent is identified, the appropriate course of treatment can be given. Initial Test Results: Gram-negative bacillus and motile

In Scenario #1 the initial test results indicate a motile Gram-negative rod. Now, if the physician remembers his microbiology he will likely consider the causative agent for abdominal pain as either Salmonella or E. coli (both are common pathogens of food-borne illnesses). However, the initial test results are not enough. The physician then orders the isolated bacterium to be streaked out on EMB agar. The next day he receives a report of a distinct metallic green growth pattern and immediately knows the causative agent is E. coli, not Salmonella.

Scenario #2 Two different work crews are in charge of cleaning your workplace. Company 1 is responsible for cleaning the bathroom sinks and toilets, while Company 2 is responsible for cleaning the floors. Several office workers have been recently treated for a Staph aureus infection, and it is believed one of the cleaning crews did not follow the proper sanitary guidelines while cleaning. The company/source responsible needs to be quickly identified and dealt with before more people become exposed. Samples are collected from various areas around the building, as well as from the cleaning tools (mops, sponges, etc.) to test whether the same or different pathogen is present throughout the workplace. Initial Test Results:Company 1 & 2: Gram-positive cocci* and non-motile for all samples* Test results were inconclusive for the presence of clusters or chains of cocci In both scenarios one of the most important things to realize is that the samples are being harvested from within a diverse environment—often many different types of microorganisms are present within the same area being sampled—some are relevant to the disease, while most are not. Therefore, the goal is to isolate and grow the potentially pathogenic species while excluding the non-pathogenic bacteria. As we discussed at the beginning of this module, often times expanding a given microbial population is required to assess the microorganism more easily. Since the causative agent in both scenarios above is unknown and likely in small amounts, the first step is to expand any potential microbial populations. The easiest way to do this is to inoculate a simple growth media with the collected sample or streak it onto an agar plate. The culture (or plate) is then grown at 37°C to encourage microbial growth. Once a bacterial population has been expanded, samples can be taken and re-streaked onto selective and/or differential media for further analysis.

In Scenario #2, although workers were already being treated for Staph aureus it is unclear if one, both, or neither of the cleaning companies were responsible for its spread. Initial test results indicated both companies could potentially be the source as both sets of samples contained non-motile, Gram (+) cocci—key characteristics of Staph aureus. Unfortunately, the test results came back with an inconclusive result regarding the exact morphology of the bacteria isolated from each company. (As a trained microbiologist would know, if any results came back as decisively Gram-positive cocci chains, this immediately rules out Staph aureus as it forms Gram-positive clusters). As an alternative strategy, a researcher then streaks the isolated bacterial samples from each company onto MSA agar. After incubating the samples overnight at 37°C, Company 1 samples showed only colorless colonies growing on red agar indicating the presence of non-pathogenic bacteria, while the samples collected from Company 2 (floors and their cleaning mops and sponges) showed a distinct yellow agar indicating the presence of Staph aureus. Obviously, the two scenarios described above are not all-inclusive examples of the pathogenic (disease-causing) effects of microorganisms. However, they do serve as a general outline for how to approach unknown samples. As will be discussed in the next module, it is a diverse microbial world, and, unfortunately, many microbes are also pathogenic.

What is agar?

In its simplest form agar is liquid growth media that has had a hardening agent added to it. Agar is created when a polysaccharide derived from seaweed (algae) extract is added to growth media. Agar is used to create a solid, smooth surface on which microbes can grow.

In a three-phase dilution gradient, which phase most likely contains individual colonies: phase 1 (P1) or phase 3 (P3)? Your Answer:

Individual colonies are most likely going to appear within the phase 3 streaks. You begin with P1 (highest concentration), dilute during P2 and then further dilute the sample in P3. However, it is possible to see individual colonies in P2 if only a small portion of P1 was carried over into P2.

LB Agar

LB agar, also known as lysogeny broth, is a multi-purpose media capable of growing a wide variety of microorganisms. LB agar plates are pale yellow in color as shown in Figure 4.1. LB is classified as a non-selective and non-differential media. LB agar is commonly used for growing E. coli, a microbe often associated with molecular microbiology applications based on its ability to easily produce recombinant proteins. Figure 4.1. Agar Plates. LB agar plates are shown with either the top lid in place (left) or tilted off (right). The agar is melted, sterilized, and poured into a sterile petri dish then allowed to solidify at room temperature.

Name the type of plate derived from BAP that contains lysed red blood cells.

Lysed red blood cells (RBCs) are a primary component in the formulation of Chocolate agar.

MacConkey Agar

MacConkey agar is pale red in color and is both a selective and differential media. The presence of crystal violet and bile salts in its formulation restricts Gram-positive bacterial growth. As such, only Gram-negative microbes can be grown on MacConkey agar. Further, lactose and the pH indicator neutral red—it is only red when under acidic conditions—are added to differentiate between lactose fermenters (lac+; red colonies) and non-fermenters (lac-; white/tan colonies). The base color of the agar plate also often changes color as the microbes consume the nutrients within the agar. The agar can turn yellow (pH > 8.0) within the surrounding area of a growing non-fermenting microbe. Similarly, the agar may turn pink or even a darker red within the region surrounding a lac+ microbe given its level of acid production during fermentation (pH < 6.8). For example, the Gram-negative/lac+ bacteria Escherichia coli is often used to demonstrate the formation of vibrant pink colonies. Within the clinical laboratory setting, where each patient sample may contain hundreds of species of bacteria, MacConkey agar is used to isolate intestinal pathogenic microbes belonging to the Enterobacteriaceae family such as Salmonella and Shigella species.

Define the selective and differential abilities of a MacConkey agar plate.

MacConkey agar is selective in that only Gram-negative microbes will grow on the agar—Gram-positive microbes simply do not grow. MacConkey agar is also considered a differential media as it distinguishes between microbes capable of fermenting lactose (red colonies) and those that are non-fermenters (white/tan colonies).

Mannitol Salt Agar

Mannitol salt agar, also known as MSA, is red in color and is both a selective and differential media. MSA is selective for Gram-positive bacteria and can differentiate members of the Staphylococci family. MSA contains a high concentration of sodium chloride (~7.5-10%) that prevents the growth of other microorganisms (selectivity) while the presence of an alternate sugar source, mannitol, along with the pH indicator dye phenol red serves to differentiate pathogenic from non-pathogenic strains of Staph. Pathogenic strains, such as Staphylococcus aureus, have the ability to ferment mannitol, thus lowering the pH of the medium and changing the dye color from red (pH >8.2) to yellow (pH < 6.8). To illustrate the similarities and differences in agar medias, a collective and generalized representation can be seen in Figure 4.2. Noting the color differences and the associated properties of each plate will be essential for correctly identifying an unknown pathogen.

Note

Note: Outside of the lab you've probably come across the cousin of agar, gelatin. Gelatin is an animal-derived product that gives Jell-O its classic texture and form. Agar is simply the plant-based form and acts in a very similar way. In both cases, as you increase the amount of agar/gelatin, the firmer the composition becomes. The dehydrated agar (and other necessary components) is mixed with water and heated to a high temperature to dissolve the agar into solution. The high temperature also ensures sterility as it will kill most, if not all, foreign microbes. Once poured into a sterile petri dish and cooled, the agar plate is a sterile environment on which microbes can grow.

Note

Note: Two common deviations to the four-phase dilution streak method involves either fewer phases (most often three-phases instead of four) or passing the sterile loop through the previous quadrant multiple times (instead of only once) when streaking out the next phase. Most often a reduction in phases or an increase in the number of times the sterile loop enters the previous phase is dependent upon the starting concentration of the sample. For example, if the bacteria are slow growing a researcher may opt to only dilute the sample into three phases. Further, greater growth can be encouraged by passing the sterile loop through the previous phase multiple times. Taken together, it would not be surprising to see various combinations of a dilution streak plate within real-world settings (i.e. a 3-phase with multiple passes vs. a 3-phase with single passes between phases vs. a 4-phase single (or multiple) passes, etc.). Either deviation is acceptable in practice provided the resulting gradient contains within it the growth of individual colonies—if not, the experiment must be repeated.

Plating

Now that the various types of growth media and nutrient agars have been outlined, the question remains: How are these materials used to grow microorganisms? The process of spreading a bacterial culture onto a petri dish filled with agar is called plating. Plating can be done using a sterile loop, a sterile swab, or a sterilized wire loop. Each device is simply a means of spreading the bacteria, most commonly in a simple back-and-forth motion, across the plate. Note: The process of plating will be visually demonstrated in the lab section of this course. The primary advantage of plating a bacterial sample onto agar is that cells are held in place. Unlike in a nutrient broth where bacterial cells can multiply but are free to move around in solution, bacteria plated onto agar are fixed in such a way as to support the formation and visualization of colonies. Colonies are visible to the naked eye but only after the bacterial cell has multiplied, often a million times over. Thus, each colony is derived from a single cell and should be free from outside contaminants (other microbes). The process of obtaining an individual colony (as opposed to a lawn growth pattern) will be discussed in the next section.

Bacterial Isolation in Practice

Now that we've covered the various types of plates on which microbes can grow and how we then go about obtaining and isolating a pure culture, the question remains: Why would a researcher ever want to do so? Let's look at some real-world situations:

Figure 4.3. Agar Streak. A 4-phase dilution streak is shown beginning with the first phase (P1) and ending with the fourth phase (P4). A sterile loop must be used when switching between phases in order to accurately generate a dilution series. Single colonies should be evident within P3 or P4.

Once the samples are appropriately streaked onto the media (and the lids placed back onto the plate), they are inverted to prevent any potential contaminants from settling onto the surface of the agar and placed in an incubator set at 37°C for ~12-24 hours. Maintaining a temperature of 37°C is optimal for promoting growth and is the temperature most commonly used by researchers. Following an overnight (>12 hours) incubation, the regions within each phase (P1 through P4) are examined and individual colonies identified. Individual colonies are then isolated, picked, and re-inoculated in nutrient broth (or onto nutrient agar) for the expansion of the now pure culture to be used for laboratory testing. Note: Alternative incubation temperatures may be used. Unlike bacteria, other organisms such as yeast preferentially grow at 30°C. Additionally, because pathogenic strains of bacteria tend to grow faster than non-pathogenic strains at 37°C, researchers may set incubators at 25°C to restrict pathogenic growth.

What is the name of the process of spreading a bacterial culture onto a petri dish?

Plating. Plating microbes can be done using a sterile loop, a sterile swab, or a sterilized wire loop. Each device is simply a means of spreading the bacteria, most commonly in a simple back-and-forth motion, across the plate.

Selective Media

Selective media allows for only the growth of certain microbes and, by extension, restricts the growth of all others. Such an approach can be accomplished in a variety of ways including using limiting amounts of nutrients, varying degrees of pH (being either very acidic or very basic media), or various chemical additives that limit unwanted microbial growth (i.e. antibiotics). Selective media is often used in medical laboratories for the cultivation of human pathogens such as Neisseria meningitides, the potential causative agent of meningitis. Because Neisseria meningitides is a relatively slow-growing organism, other bacteria, mold, or fungi often outgrow the sample—remember medical samples often contain numerous unknown microbes! —therefore, additives are included in the growth media to specifically inhibit such foreign microbes and ensure the isolated growth of Neisseria meningitides.

What is selective media?

Selective media allows for only the growth of certain microbes while restricting the growth of all others.

Sorbitol-MacConkey Agar

Sorbitol-MacConkey agar, also known as SMAC, is a variant of MacConkey agar specifically formulated to detect the presence of the pathogenic strain of Escherichia coli O157:H7. Under standard enteric (gut) conditions, Escherichia coli is able to ferment both lactose and sorbitol. However, E. coli O157:H7 is unable to ferment sorbitol. Therefore, SMAC plates contain sorbitol (instead of lactose) in order to differentiate non-pathogenic strains of Escherichia coli (red colonies; acidic conditions) from pathogenic O157:H7 strains (white colonies; neutral to basic conditions).

What colors would you expect to see on an EMB plate containing E. coli?

The EMB plate itself is red in color while in the presence of E coli, the growing colonies will take on a distinctive metallic green sheen. Note: You are responsible for knowing the color and general properties (selective vs differential) of all of the agar plates described within this module, with a special emphasis on how a particular agar plate aids in identifying select microbes.

What is the primary advantage of plating a bacterial culture as opposed to having it grow in solution?

The primary advantage of plating a bacterial sample onto agar is that cells are held in place. Unlike in a nutrient broth where bacterial cells can multiply but are free to move around in solution, bacteria plated onto agar are fixed in such a way as to support the formation and visualization of colonies.

What is the purpose of the quadrant streak approach?

The purpose of this method is to generate an individual colony so that a single (pure) bacterial sample can be picked from the plate.

Obtaining a Pure Culture

To isolate a microbe on an agar plate, microbiologists often utilize a quadrant streak (or phase-dilution) approach. The purpose of this method is to generate an individual colony so that a single (pure) bacterial sample can be picked from the plate. Importantly, a pure culture is free of outside contaminants and can be traced back to a single origin. Once isolated and expanded, a pure culture can be further examined for its size and shape, motility, Gram status, biochemical properties, etc. As outlined below, the sample is spread across the plate in such a way as to establish a dilution gradient. The resulting gradient should always contain within it the growth of individual colonies. For a four-phase dilution gradient, the sample is spread into four regions where each new region is perpendicular to the previous region, such that each region will become more diluted than the previous. As shown in Figure 4.3 and staying within the first phase (P1), a sterile loop is first used to streak a sample back-and-forth across a small area of the plate. The first region will contain the highest concentration of microbial growth. Next, a new sterile loop will be used to pull some of the sample from the first region into the second phase (P2), where it is further streaked. The microbial concentration is now diluted because only a small portion of the bacterial sample from phase one was moved and spread out into P2. The same process is repeated from P2 to P3, and then from P3 to P4, each time using a new sterile loop. Thus, each phase should be systematically diluted such that the bacterial concentration of each phase will be established as P1 > P2 > P3 > P4. As the process is carried out, a new (or sterilized) loop must be used each time a new phase is started. Failure to do so would prevent the establishment of a dilution gradient, as the same bacterial concentration would be spread across both phase regions. Again, each subsequent region (phase) should contain a lower concentration of microbes such that an individual colony will be eventually produced. Although individual colonies are most often seen in P4, depending on the starting concentration in P1 individual colonies may also begin to appear in P3 or even in P2.

Trypticase Soy Agar

Trypticase soy agar, also known as TSA or TSAYE, is another multi-purpose media capable of growing a wide variety of microorganisms. TSA plates are virtually identical in color to LB plates. TSA media is non-selective and non-differential and serves as the base when formulating specialized (enriched) medias.