Microbial Cultures, Biofilms, and Safety

Hi, my name is Dr. Alice Lee, and in this lecture we will be talking about the importance of pure cultures, what biofilms are, and the various biosafety levels in which we operate to ensure the safety of our lab personnel when working with microbial cultures. This image is from the CDC website showing a lab worker in his positive pressure suit being decontaminated in a chemical shower after working in a biosafety level 4 lab. CDC is one of only a handful of facilities in the United States with biosafety levels. 4. Laboratory space capable of handling contagions for which there's no treatment or vaccine. Yeah, I'm not sure I'd be brave enough to work in ABSL 4. Now this guy, he's brave. So bacteria are everywhere. On benchtops, in water, soil, and food. On your skin, in your ears, nose, throat, and intestinal tract. which they're considered all our normal flora. The diversity of bacteria present in our environment and in our bodies is incredible. When trying to study bacteria from the environment, you would discover that bacteria usually exist in mixed populations. It is only in very rare situations that bacteria can occur as a single species. However, to be able to study the cultural, morphological, and physiological characteristics of an individual species, it is essential that the organism be separated from other species that are normally found in its habitat. In other words, we must establish what is called a pure culture of the microorganism. A pure culture is defined as a population containing only a single species or strain of bacteria. A culture is considered a pure culture if only one type of organism is present. and it's considered a mixed culture if populations of different organisms are present. As seen here on this top plate, note the different colony types. When first used, the culture medium should also be sterile, meaning that no form of life is present before inoculation with the microbe. Contamination means that more than one species is present in a culture that's supposed to be pure. Contamination does not imply that the contaminating organism is harmful. It simply means that the contaminating organism is unwanted in the culture that you're trying to isolate and study. Special techniques, commonly referred to as aseptic pure culture techniques, must be used to obtain a single isolated strain for study. Therefore, pure culture technique allows large populations of essentially identical organisms to be prepared, free of other microbes. It's important to note that a pure culture bacteria is ideally derived from a single cell But in practice, it starts with a small group of cells. Given the exponential reproduction rate and the rate of spontaneous mutations, even a pure bacterial culture will contain some spontaneous mutant cells and that will vary in their nucleotide sequences from the original cells. Thus the culture is not perfectly homogenous. Now a colony is a visible mass of microbial cells arising from one cell or spore or from a group of the same microbes. Often though, two or more cells are in a clump and they will grow to form only a single colony. So if a sample contains many clumps, a viable count, viable here means that it successfully replicates to form a colony, of that sample may be erroneously low. Data from such samples are often expressed as what we call colony forming units that's obtained rather than the actual number of viable cells because a colony forming unit may contain one or more cells. Thus, each colony on an agar plate represents one viable organism, or CFU, present in the original liquid culture. Most bacteriological work requires pure cultures or clones of bacteria. The isolation method most commonly used to get pure cultures is the streak plate method. It's also known as the T-streak, the quaggion streak, the isolation streak. In the streak plate method, a small amount of inoculum is picked up on a sterile inoculating loop made of wire. It's easily sterilized generally by heating. The loop is drawn repeatedly across the surface of the plate diluting microbes as it moves. Here in figure 6.11 arrows indicate the direction of streaking with an inoculating loop. Streak series 1 is made from the original bacterial culture. The inoculating loop is sterilized following each streak series. In series 2 and 3, the loop picks up bacteria from the previous series. diluting the number of cells each time. By series 3, notice that the well-isolated colonies of bacteria here in this plate on the right, there are some red colonies and some yellow colonies that have been obtained. So in nature, microbes seldom live in isolated single species colonies that we see on our lab plates. They more typically live in communities that we call biofilms. Biofilms usually consist of a mixed bacterial population, but they may also consist of just a single bacterial species. Some biofilms contain bacteria, viruses, and even eukaryotic cell types, most often yeast. These biofilm communities consist of what we call sessile organisms, meaning that they grow attached to a surface. Biofilms on surfaces have a characteristic structural consisting of micro colonies enclosed in a hydrated matrix of microbially produced proteins nucleic acids and polysaccharides often this is also called a slime or a hydrogel in this complex biofilm network the cells act less as individual entities and more as a collective living system often with channels to deliver water and nutrients to the cells at the inner portion of the biofilm Biofilm organisms are significantly more resistant to environmental stresses or microbially deleterious substances such as antibiotics and biocides than their planktonic or floating or free-living type cells. Biofilm cells present on infected tissues or medical devices are less susceptible to host immune responses than planktonic cells. The development of a biofilm in vitro involves the following five stages. Stage 1. always includes the reversible attachment of bacterial cells to a surface. Stage 2, irreversible attachment is mediated by formation of an exopolymeric, usually made of polysaccharides, nucleotides, things like that, material. Stage 3 is formation of these micro colonies and the beginning of biofilm maturation. In stage 4, formation of mature biofilm with it. Very distinct three-dimensional structure containing cells packed in clusters with channels between the clusters will allow for transport of water and nutrients and waste removal. And in stage five, what occurs sometimes is detachment and dispersal of cells from the biofilm and initiation of new biofilm formation. Dispersed cells are more similar to planktonic, that is these non-adherent free-floating, energy-living cells. than to the mature biofilm cells. Oftentimes, the bacteria that become part of a biofilm engage in a quorum sensing type of decision-making process in which their behavior is coordinated through a chemical vocabulary. Although the mechanisms behind quorum sensing are not fully understood, the communication process allows, for example, a single cell bacteria to perceive how many other bacteria are in close proximity. If a bacterium can sense that it's surrounded by a dense population of other pathogens, it's more likely to be inclined to join them and then contribute to formation of a biofilm. There are a few advantages to the growth pattern of biofilms. First, bacteria are protected from the inhibitory effects of antimicrobial compounds, biocides, chemical stresses such as pH and oxygen, and physical stresses like pressure, heat, and freezing. Secondly, the polymeric matrix increases the binding of water and leads to a decreased chance of dehydration of the bacterial cells. This is a stress that planktonic cells are often subject to. And third, close proximity to microbes in a biofilm allows nutrients, metabolites, and genetic material to be readily exchanged. So microbes in a biofilm can work cooperatively to carry out very complex tasks. For example, the digestive system of ruminant animals, such as cattle, require many different species to break down cellulose, most of the microbes are in a biofilm that line its digestive tract. This probably works the same way in your digestive tract. Oftentimes though, biofilms are an important factor in health. For example, microbes in a biofilm were found to be a thousand times more resistant to antibiotics. According to a recent public statement from the National Institutes of Health, more than 70 percent of all microbial infections are caused by biofilms. Think urinary tract infections or UTIs. periodontitis, ear infections, chronic wounds, cystic fibrosis, chronic sinusitis. One study found that biofilms are present on the removed tissue of two-thirds of patients undergoing surgery for chronic inflammation of the sinuses. The development of a biofilm allows for the cells inside to become much more resistant to the body's natural antimicrobials as well as the antibiotics, causing antibiotic resistance when it is administered in a standard fashion. Biofilms can also be a problem in industry. They can be a problem in pipes and tubing where their accumulations impede circulation, such as in indwelling medical devices, including mechanical heart valves and catheters. Biofilms can also do long-term damage to water distribution facilities and other public utilities, causing fouling of equipment and contamination of products. The study of biofilms is thus an important aspect of medical and industrial microbiology. So, handling all these microbes requires specialized laboratory facilities and techniques. Biosafety is the application of safety precautions that reduce a scientist's risk of exposure to potentially infectious microbes and also limits the contamination of the work environment. We designate Moosabab labs under four special hazard categories called biosafety levels or BSLs. Each level has specific controls for the containment of microbes and biological agents. Each biosafety level builds on the controls before it. All of these levels followed what we call standard microbiological practices, which are those practices common to all labs, which include not eating or drinking or applying cosmetics, washing hands after working in the lab, and routinely decontaminating the work area. So, in the BSL-1 labs, if you work in a lab that's designated as a BSL-1, the microbes there are not known to consistently cause disease in healthy adults and present disease. minimal potential hazard to laboratorians and the environment. An example of a microbe that's typically worked with at BSL-1 is a non-pathogenic strain of E. coli. Work can be done on an open lab bench and it requires a sink. It generally requires personal protective equipment or PPE, such as lab coats, gloves, and eye protection. BSL-2 builds on BSL-1. If you work in a lab that's designated BSL-2, the microbes there pose moderate hazards to laboratorians and the environment. The microbes are typically indigenous and associated with diseases of varying severity. An example of a microbe that's typically worked with in a BSL-2 laboratory is Staphylococcus aureus. It includes various bacteria and viruses that cause only mild disease in humans or they're difficult to contract via aerosols in a lab setting such as Clostridium difficile, Moschlemidi, hepatitis A, B, and C, influenza viruses, Salmonella. The BSL-2 differs from BSL-1 in that laboratory personnel generally have specific training in handling pathogenic agents and they are directed by scientists with advanced training. Access to the lab is limited when work is being conducted, extreme precautions are taken with contaminated sharp items, and certain procedures in which infectious aerosols or splashes may be created or conducted inside a biosafety cabinet or other physical containment equipment. BSL-3 is required to work when involving indigenous or exotic agents that can cause serious or potentially lethal disease that are transmitted through the air or via aerosols. Respiratory transmission is the inhalation route of exposure. Laboratory personnel must receive specific training in handling pathogenic and potentially lethal agents and they must be supervised by scientists competent in handling infectious agents and associated procedures. Lab personnel are often under medical surveillance and might receive immunization for microbes they work with. All procedures involving the manipulation of infectious materials must be conducted within biosafety cabinets, shown at the top image here, or it has to be performed on their other physical containment devices, and the personnel has to be wearing appropriate PPE and sometimes even respirators. A BSL-3 laboratory has special engineering and design features that prevent the release of microbes to the environment. Facilities have a hands-free sink, exhaust air cannot be recirculated, entrances through two sets of self-closing and locking doors. Microbes that are worked on in BSL-3 facilities are mycobacterium tuberculosis, which causes tuberculosis, for example, and other organisms. BSL-4 labs builds on the containment requirements. requirements of BSL-3 and is the highest level of biological safety. is required for work with dangerous and exotic agents that pose a high individual risk of life-threatening disease. Aerosol transmission or unknown risk of transmission also. The microbes in these BSL-4 labs cause infections that are frequently fatal, and generally there are no vaccines or treatments for these infections. Note there are only a small number of such labs in the U.S., probably less than 10, and also in the world. Laboratory staff must have specific and thorough training in handling extremely hazardous infectious agents. Access to the laboratory is controlled by the laboratory supervisor and all handling of agents must be performed in a gas-tight class 3 biosafety cabinet or by personnel wearing positive pressure protective suits that kind of look like spacesuits. The BSL-4 labs have special engineering design features to prevent microbes from being released in the environment. The labs usually in a separate building or isolated restricted zone of the building. It also has a dedicated supply and exhaust air as well as vacuum lines and decontamination systems. Personnel must change clothing before entering and shower upon exiting. Most of the pathogens worked on here are viruses such as the cremaine, congo, hemorrhagic fever caused by strains such as ebola, junen, lassa, machupu, and marlboro viruses, tick-borne encephalitis virus complex which includes a long list of viruses. Hey, it's me down here. Someone labeled my coffee mug with a biohazard symbol. So the suggested reading for this particular lecture is chapter 6 again in your tutorial text. Here's a picture of me actually holding up a pure culture streak plate of Serratia marcescens. It's a common environmental bacterium that produces a red pigment called prodigiosin. We use this particular microbe quite frequently in the teaching labs.

So bacteria are everywhere. On benchtops, in water, soil, and food. On your skin, in your ears, nose, throat, and intestinal tract.

which they're considered all our normal flora. The diversity of bacteria present in our environment and in our bodies is incredible. When trying to study bacteria from the environment, you would discover that bacteria usually exist in mixed populations.

It is only in very rare situations that bacteria can occur as a single species. However, to be able to study the cultural, morphological, and physiological characteristics of an individual species, it is essential that the organism be separated from other species that are normally found in its habitat. In other words, we must establish what is called a pure culture of the microorganism.

A pure culture is defined as a population containing only a single species or strain of bacteria. A culture is considered a pure culture if only one type of organism is present. and it's considered a mixed culture if populations of different organisms are present. As seen here on this top plate, note the different colony types. When first used, the culture medium should also be sterile, meaning that no form of life is present before inoculation with the microbe.

Contamination means that more than one species is present in a culture that's supposed to be pure. Contamination does not imply that the contaminating organism is harmful. It simply means that the contaminating organism is unwanted in the culture that you're trying to isolate and study.

Special techniques, commonly referred to as aseptic pure culture techniques, must be used to obtain a single isolated strain for study. Therefore, pure culture technique allows large populations of essentially identical organisms to be prepared, free of other microbes. It's important to note that a pure culture bacteria is ideally derived from a single cell But in practice, it starts with a small group of cells.

Given the exponential reproduction rate and the rate of spontaneous mutations, even a pure bacterial culture will contain some spontaneous mutant cells and that will vary in their nucleotide sequences from the original cells. Thus the culture is not perfectly homogenous. Now a colony is a visible mass of microbial cells arising from one cell or spore or from a group of the same microbes. Often though, two or more cells are in a clump and they will grow to form only a single colony.

So if a sample contains many clumps, a viable count, viable here means that it successfully replicates to form a colony, of that sample may be erroneously low. Data from such samples are often expressed as what we call colony forming units that's obtained rather than the actual number of viable cells because a colony forming unit may contain one or more cells. Thus, each colony on an agar plate represents one viable organism, or CFU, present in the original liquid culture.

Most bacteriological work requires pure cultures or clones of bacteria. The isolation method most commonly used to get pure cultures is the streak plate method. It's also known as the T-streak, the quaggion streak, the isolation streak. In the streak plate method, a small amount of inoculum is picked up on a sterile inoculating loop made of wire.

It's easily sterilized generally by heating. The loop is drawn repeatedly across the surface of the plate diluting microbes as it moves. Here in figure 6.11 arrows indicate the direction of streaking with an inoculating loop.

Streak series 1 is made from the original bacterial culture. The inoculating loop is sterilized following each streak series. In series 2 and 3, the loop picks up bacteria from the previous series. diluting the number of cells each time. By series 3, notice that the well-isolated colonies of bacteria here in this plate on the right, there are some red colonies and some yellow colonies that have been obtained.

So in nature, microbes seldom live in isolated single species colonies that we see on our lab plates. They more typically live in communities that we call biofilms. Biofilms usually consist of a mixed bacterial population, but they may also consist of just a single bacterial species. Some biofilms contain bacteria, viruses, and even eukaryotic cell types, most often yeast. These biofilm communities consist of what we call sessile organisms, meaning that they grow attached to a surface.

Biofilms on surfaces have a characteristic structural consisting of micro colonies enclosed in a hydrated matrix of microbially produced proteins nucleic acids and polysaccharides often this is also called a slime or a hydrogel in this complex biofilm network the cells act less as individual entities and more as a collective living system often with channels to deliver water and nutrients to the cells at the inner portion of the biofilm Biofilm organisms are significantly more resistant to environmental stresses or microbially deleterious substances such as antibiotics and biocides than their planktonic or floating or free-living type cells. Biofilm cells present on infected tissues or medical devices are less susceptible to host immune responses than planktonic cells. The development of a biofilm in vitro involves the following five stages. Stage 1. always includes the reversible attachment of bacterial cells to a surface.

Stage 2, irreversible attachment is mediated by formation of an exopolymeric, usually made of polysaccharides, nucleotides, things like that, material. Stage 3 is formation of these micro colonies and the beginning of biofilm maturation. In stage 4, formation of mature biofilm with it.

Very distinct three-dimensional structure containing cells packed in clusters with channels between the clusters will allow for transport of water and nutrients and waste removal. And in stage five, what occurs sometimes is detachment and dispersal of cells from the biofilm and initiation of new biofilm formation. Dispersed cells are more similar to planktonic, that is these non-adherent free-floating, energy-living cells. than to the mature biofilm cells. Oftentimes, the bacteria that become part of a biofilm engage in a quorum sensing type of decision-making process in which their behavior is coordinated through a chemical vocabulary.

Although the mechanisms behind quorum sensing are not fully understood, the communication process allows, for example, a single cell bacteria to perceive how many other bacteria are in close proximity. If a bacterium can sense that it's surrounded by a dense population of other pathogens, it's more likely to be inclined to join them and then contribute to formation of a biofilm. There are a few advantages to the growth pattern of biofilms.

First, bacteria are protected from the inhibitory effects of antimicrobial compounds, biocides, chemical stresses such as pH and oxygen, and physical stresses like pressure, heat, and freezing. Secondly, the polymeric matrix increases the binding of water and leads to a decreased chance of dehydration of the bacterial cells. This is a stress that planktonic cells are often subject to. And third, close proximity to microbes in a biofilm allows nutrients, metabolites, and genetic material to be readily exchanged.

So microbes in a biofilm can work cooperatively to carry out very complex tasks. For example, the digestive system of ruminant animals, such as cattle, require many different species to break down cellulose, most of the microbes are in a biofilm that line its digestive tract. This probably works the same way in your digestive tract.

Oftentimes though, biofilms are an important factor in health. For example, microbes in a biofilm were found to be a thousand times more resistant to antibiotics. According to a recent public statement from the National Institutes of Health, more than 70 percent of all microbial infections are caused by biofilms.

Think urinary tract infections or UTIs. periodontitis, ear infections, chronic wounds, cystic fibrosis, chronic sinusitis. One study found that biofilms are present on the removed tissue of two-thirds of patients undergoing surgery for chronic inflammation of the sinuses. The development of a biofilm allows for the cells inside to become much more resistant to the body's natural antimicrobials as well as the antibiotics, causing antibiotic resistance when it is administered in a standard fashion.

Biofilms can also be a problem in industry. They can be a problem in pipes and tubing where their accumulations impede circulation, such as in indwelling medical devices, including mechanical heart valves and catheters. Biofilms can also do long-term damage to water distribution facilities and other public utilities, causing fouling of equipment and contamination of products. The study of biofilms is thus an important aspect of medical and industrial microbiology.

So, handling all these microbes requires specialized laboratory facilities and techniques. Biosafety is the application of safety precautions that reduce a scientist's risk of exposure to potentially infectious microbes and also limits the contamination of the work environment. We designate Moosabab labs under four special hazard categories called biosafety levels or BSLs.

Each level has specific controls for the containment of microbes and biological agents. Each biosafety level builds on the controls before it. All of these levels followed what we call standard microbiological practices, which are those practices common to all labs, which include not eating or drinking or applying cosmetics, washing hands after working in the lab, and routinely decontaminating the work area. So, in the BSL-1 labs, if you work in a lab that's designated as a BSL-1, the microbes there are not known to consistently cause disease in healthy adults and present disease.

minimal potential hazard to laboratorians and the environment. An example of a microbe that's typically worked with at BSL-1 is a non-pathogenic strain of E. coli. Work can be done on an open lab bench and it requires a sink.

It generally requires personal protective equipment or PPE, such as lab coats, gloves, and eye protection. BSL-2 builds on BSL-1. If you work in a lab that's designated BSL-2, the microbes there pose moderate hazards to laboratorians and the environment.

The microbes are typically indigenous and associated with diseases of varying severity. An example of a microbe that's typically worked with in a BSL-2 laboratory is Staphylococcus aureus. It includes various bacteria and viruses that cause only mild disease in humans or they're difficult to contract via aerosols in a lab setting such as Clostridium difficile, Moschlemidi, hepatitis A, B, and C, influenza viruses, Salmonella. The BSL-2 differs from BSL-1 in that laboratory personnel generally have specific training in handling pathogenic agents and they are directed by scientists with advanced training.

Access to the lab is limited when work is being conducted, extreme precautions are taken with contaminated sharp items, and certain procedures in which infectious aerosols or splashes may be created or conducted inside a biosafety cabinet or other physical containment equipment. BSL-3 is required to work when involving indigenous or exotic agents that can cause serious or potentially lethal disease that are transmitted through the air or via aerosols. Respiratory transmission is the inhalation route of exposure.

Laboratory personnel must receive specific training in handling pathogenic and potentially lethal agents and they must be supervised by scientists competent in handling infectious agents and associated procedures. Lab personnel are often under medical surveillance and might receive immunization for microbes they work with. All procedures involving the manipulation of infectious materials must be conducted within biosafety cabinets, shown at the top image here, or it has to be performed on their other physical containment devices, and the personnel has to be wearing appropriate PPE and sometimes even respirators.

A BSL-3 laboratory has special engineering and design features that prevent the release of microbes to the environment. Facilities have a hands-free sink, exhaust air cannot be recirculated, entrances through two sets of self-closing and locking doors. Microbes that are worked on in BSL-3 facilities are mycobacterium tuberculosis, which causes tuberculosis, for example, and other organisms. BSL-4 labs builds on the containment requirements.

requirements of BSL-3 and is the highest level of biological safety. is required for work with dangerous and exotic agents that pose a high individual risk of life-threatening disease. Aerosol transmission or unknown risk of transmission also. The microbes in these BSL-4 labs cause infections that are frequently fatal, and generally there are no vaccines or treatments for these infections.

Note there are only a small number of such labs in the U.S., probably less than 10, and also in the world. Laboratory staff must have specific and thorough training in handling extremely hazardous infectious agents. Access to the laboratory is controlled by the laboratory supervisor and all handling of agents must be performed in a gas-tight class 3 biosafety cabinet or by personnel wearing positive pressure protective suits that kind of look like spacesuits. The BSL-4 labs have special engineering design features to prevent microbes from being released in the environment. The labs usually in a separate building or isolated restricted zone of the building.

It also has a dedicated supply and exhaust air as well as vacuum lines and decontamination systems. Personnel must change clothing before entering and shower upon exiting. Most of the pathogens worked on here are viruses such as the cremaine, congo, hemorrhagic fever caused by strains such as ebola, junen, lassa, machupu, and marlboro viruses, tick-borne encephalitis virus complex which includes a long list of viruses. Hey, it's me down here. Someone labeled my coffee mug with a biohazard symbol.

So the suggested reading for this particular lecture is chapter 6 again in your tutorial text. Here's a picture of me actually holding up a pure culture streak plate of Serratia marcescens. It's a common environmental bacterium that produces a red pigment called prodigiosin. We use this particular microbe quite frequently in the teaching labs.

Transcript for:Microbial Cultures, Biofilms, and Safety

Transcript for:
Microbial Cultures, Biofilms, and Safety