Microbiology Report

MicrobiologyIbarra, Camille

Karunungan, JennyLevy, Daniel

Lim, JosellLomotan, AlfieMateos, Eloisa

Materum, AdrianMaturino, Kendrick

The study of microscopic organisms – any living organism that is either a single cell, cell cluster, or no celled organism.

Organisms cannot be seen with the naked eye, cannot be distinguished phylogenically from other macroorganisms.

Microbiologists investigate growth and characteristics of microscopic organisms by isolating and culturing them.

Microbiology

Existence of microorganisms was hypothesized centuries before their actual discovery.

In 1546 Girolamo Fracastoro proposed that epidemic diseases were caused by transferrable seed-like entities that could transmit infection by direct or indirect contact, or vehicle transmission.

In 1665, Robert Hooke made the first recorded microscopic observation of the fruiting bodies of molds

In 1676, Anton Van Leeuwenhoek observed bacteria and other microorganisms, using a single-lens microscope of his own design.

History of Microbiology

Late 19th century, with the work of the founders of general microbiology (Martinus Beijerinck and Sergei Winogradsky) the true breadth of microbiology was revealed.

History (cont’d)

Bacteriology - The study of bacteria. Mycology - The study of fungi. Protozoology - The study of protozoa. Phycology (or Algology) - The study of algae. Parasitology - The study of parasites. Immunology - The study of the immune system. Virology - The study of viruses. Nematology - The study of the nematodes

Branches of Microbiology

Medical MicrobiologyInvolves in identifying, treatment or prevention of some diseases caused by bacteria, virus, and fungi. The medical microbiologists research on different diseases, their causes, development and pathogenesis.

Food MicrobiologyIt deals with the fermentations and fermented products like yogurt, beer, wine etc. These microbiologists involve in prevention of deterioration and spoilage of the food products and have a continuous hygiene checks on the food products to prevent any food poisonings.

Environmental MicrobiologyIt involves in the study of ecological study which includes air, water, food and environment as such. These microbiologists check the factory wastes which can cause pollution of the air or water thus increasing the rate of poisoning or diseases in the environment.

Agricultural MicrobiologyIt is dealing with the various aspects of agriculture. Microbes act as fertilizers as they fix nitrogen in soil but some microbes attack these agricultural fields which may in-turn lead to food poisoning, when taken raw. These microbiologists deal with checking out the safety of the agricultural fields.

A Microbiology Laboratory is a unique environment that requires special practices and containment facilities in order to properly protect persons working with microorganisms. Safety in the laboratory is the primary concern. The three main elements of safe containment of microorganisms are good laboratory practices and technique, safety equipment, and facility design.

Microbiology Laboratory

Microbiology Lab Tests

1. Aseptic Technique– insures that no contaminating organisms are introduced into cultured

materials when the latter are inoculated or handled in some manner

2. Pure Culture Techniques- contains only a single kind of an organism where as a mixed culture

contains more than one kind of organisms.- able to study the cultural, morphological and physiological

characteristics of an individual organisms.-Streak plate needs more skill for economy use (material and time)

-Pour plate less skill requires more media, tubes and plates

A. MANIPULATION OF MICROOGRANISMS

Its success depends upon the preparation of a good smear

A good smear must: Cause the cells to adhere to the microscope slide so that

they are not washed off during subsequent staining and washing procedures.

Insure that shrinkage of cells does not occur during staining, otherwise distortion and artifacts can result.

Prepare thin smear because thickness of the smear will determine if you can visualize individual cells, their arrangement or details regarding microstructures associated with cells.

STAINING OBSERVATION OF MICROORGANISMS

* Preparing bacteriological smear differs according to the source of the organisms

Growing in a liquid medium – directly on the slide Solid Media – dispersed in to a water

* The most difficult concept for students to understand about making slides from solid media is that it takes only a VERY SMALL AMOUNT of material to make a good smear

Criteria of a good smear:

1. Simple Staining Use a single stain to color a bacterial cell Commonly used dye: methylene blue, basic fuchsia, crystal-

violet (BASIC DYES) All of these work well on bacteria because they have color-

bearing ions that are positively charged, thus bacteria are negatively charged.

ACIDIC DYES-those that have anionic chromophoresEx. Eosin

-will not stain bacteria (repel)

Types of Staining:

Useful in determining basic morphology and the presence / absence of certain kinds of granules.

It is good for 30sec. – 2mins (depends on the affinity of the dye)

After washing off the stain it is blotted dry by bibulous paper and examined directly under oil immersion

Simple Staining (cont’d)

2. Negative Staining Useful in studying the morphology of bacterial cells and

characterizing some of the external structures(capsule) Accurately determines cell dimension Acidic and thus have negatively charged chromophores

that does not penetrate the cell but rather is repelled by similarly charged bacterial cell.

The background surrounding the cell is colored by a negative stain, resulting indirect staining of the cell; cells appear as transparent objects against a dark background.

Dyes used: india ink , nigrosin Cells are not usually heat-fixed

Types of Staining (cont’d)

3. Capsular Staining

Capsule/glycocalyx – an extracellular slime layer of some bacteria cells ; play a protective role

Cells are not heat-fixed (cause shrinkage) Procedure: The smear is air-dried Stain with crystal violet for 2mins Stain is washed off with 20% copper sulfate solution and blotted

dry Capsules will appear as a light blue and cells will appear as dark

purple.


4. Gram Staining (Christian Gram) differential staining

*The staining reactions take advantage of the fact that cells or structures within cells display dissimilar staining reactions that can be distinguished by the use of different dyes.-useful for the identification of pathogenic bacteria2 Kinds of Cells:

-gram-positive appear purple retain a crystal violet iodine complex through decolorization with alcohol

-gram-negative appear pink to red alcohol removes the crystal violet-iodine complex counterstained with red dye(safranin)


5. Spore Staining Species that belong to Bacillus and Clostrida exhaust essential nutrients, they

undergo complex developmental cycle that produces resting stage (endospores) Allow bacteria to survive environmental conditions not favorable to their growth

◦ Has exoporium (protein coat) protective barrier around spore.◦ Heat resistant◦ Higher water = Less heat resistant

Use autoclave( has extreme heat) define conditions necessary to establish sterility

* Schaeffer – Fulton Utilizes malachite green to stain the endospore and safranin to stain vegetative

portion of cell ( If properly stained, spore former will have green endospore contained in pink sporangium)

* Dormer Produces a red spore within a colorless sporangium Used nigrosin for dark background

*Quick Spore Stain A variation of Schaeffer- Fulton method is a quick method that uses same

stain.

6. Acid- Fast Staining used to detect mycobacteria that cause tuberculosis and leprosy organisms have lipid-containing cell walls

Mycolic acid – composed of fatty acids and fatty alcohols Affect the staining properties of these bacteria and prevents them being stained by

any stains routinely used in microbiology - it has a unique characteristic in binding carbol-fuchsin stain

Ziehl - Neelsem Method “hot stain”- a carbol fuchsin contains phenol and cells are heated for 5 mins. During staining procedure.- Phenol and heat facilitate the penetration of carbolfuchsin into cell.

Modified Ziehl-Neelsen Method “cold stain”- does not use heat for fixing the primary stain

Kinyoun acid- fast method- modification in the concentration of primary stain, basic fuchsia and phenol, are increased but bacterial cells are not heated during the staining procedure.

(Acid-fast staining)

Acid-fast organisms appear red colored but when there is alcohol treatment acid-fast negative lose the color. Then when followed by couterstaining with methylene blue, the decolorized acid-fast negative organisms and other cells take the blue color and stand contrast with the red-colored acid-fast organisms


B. STERILIZATION Processes

Indicates the elimination of all viable microbes and spores

The elimination of microbiological organisms to achieve asepsis, a sterile microbial environment

Microbiology Lab Tests (cont’d)

1. Sterilization by Moist HeatA. Boiling

- Simplest method of sterilization- Boiling for 2-10 minutes will kill the vegetative forms of pathogenic

bacteria and fungi but does not kill bacterial spores of many pathogenic fungi and other viruses like the Hepatitis Virus

B. Autoclaving (Compressed Steam)- More effective than boiling- Uses steam under pressure (15lbs per square inch) with 121 degree

Celsius to kill and destroy spores more effectively- Surgical equipment and dressings, nutrient media, contaminated

specimens and others are subjected to autoclaving- Simple steam sterilization is achieved under a pressure cooker whereas

large-scale sterilization requires autoclave

Sterilization

Image of an electric boiling sterilizer

Image of a vertical autoclave

2. Sterilization by Dry Heat (Flaming or Incineration)

- This technique is only used in metals and is ideal for sterilizing loops and needles for it destroys any living forms in needles and loops

- Flame sterilization for Forceps and Scalpels are done in a Hot air oven.- Heating the Scalpel over a flame can damage its

sharpness. Preferred Heating time is only less than 2 minutes.

Sterilization (cont’d)

3. Filtration-is the main method by

which heat-liable biological fluids or liquid bacterial culture media are used to remove bacteria.

-routinely used filter are 0.2 micrometer porous disks composed of biologically inert cellular esters.


4. Sterilization by Radiation

a. Ionizing Rays-X-ray and gamma rays are much more powerful and

penetrating than ultraviolet light.-used for the sterilization of syringes, surgical gloves, plastic

items, disposable items and in the food industry. b. Ultraviolet light

- Since ultraviolet rays (260nm) in the sunlight have been found to be an effective germicide, ultraviolet light is being used to control the spread of pathogenic organisms especially in operating rooms, laboratories etc. However, it does not have any penetrating power.


C. Antimicrobial Preservative Efficacy (APE)- a test that ensures that preserved products are sufficiently

protected from microbiological contamination that has been introduced during or subsequent to the manufacturing process. A wide variety of products require APE testing including injectable drugs, toothpaste, contact lens solution and cosmetics.

- can be used alongside an accelerated shelf-life test to ensure the preservative remains stable and effective during the labeled shelf life of the product.


1. Proliferation Assay allows to determine the number of cells that are

growing in the absence or presence of certain proliferation affecting agents, e.g. TNF-alpha or anti-Fas antibody (IPO-4).

APE Continuation

PRINCIPLEA certain number of cells is seeded in the wells of a 96 well plate. At the same time either a proliferation affecting agent is added or not. The cells are incubated for a certain time (e.g. 24 h) at 37°C. Then 3H-Thymidine is added to the wells and incubated for another period of time (e.g. 24h).Cells that are incubated without any growth inhibiting agent will grow: during each cell division the cells will incorporate 3H-Thymidine into its DNA. The more cell divisions (or the higher the proliferation rate) the more radioactivity will be incorporated into DNA. Cells that are incubated in the presence of the growth inhibiting agent (e.g. TNF-alpha will incorporate less radioactivity.After incubation the cells are harvested: during harvesting, the cells are washed out of the wells of the 96 well plate with bidest water: the cells and organelles burst and the cell's DNA is set free. The cell fragments and DNA are passed through a filter membrane (glassfiber). Only particles of smaller than 1,5 µm can pass the filter. So, intact DNA (with a fragment length in the range of milimeters or even centimeters) will not be able to pass the filter but be collected on the filter membrane. The higher the proliferation rate of the cells was during incubation, the more cells are harvested and the more radioactive DNA will be collected on the filter.

2. Antibiotic assay- is used to determine the potency of the antibiotics using an assay technique. The potency of the antibiotic can be examined by creating a suitable environment. Its inhibitory effects are then studied on the respective microorganisms. An antibiotic sensitivity assay tests the efficacy of a drug in inhibiting the growth of microorganisms that causes a disease. Antibiotic assays are carried out on the infecting organism that is found in clinical specimens of body fluids such as blood and urine collected from patients seeking treatment.

3. Agar Relay Technique- This technique allows you to produce a homogeneous lawn of bacteria within a thin layer of agar across the surface of a plate. Bacteria are added to a soft top agar (0.75% agar, as opposed to the usual 1.5% for agar plates) which has been melted at 100°C and cooled to 45°C. This is warm enough so the agar remains liquid, but cool enough so that the bacteria are not killed (for a period of time). The melted agar/bacterial suspension is mixed and poured evenly across the top of an agar plate and allowed to solidify.- The bacteria distributed through the top agar will grow to produce a homogeneously turbid lawn. If the freshly seeded lawn is exposed to various antibacterial agents and then incubated at 37°C, any inhibition of bacterial growth will cause a reduction in the turbidity of the lawn near the agent: the greater the antibacterial action, the wider the zone of inhibition. Thus, the antibacterial strength of the agent may be judged by the width of the zone of inhibition around it.

D. Evaluation of Biological Indicatiors

Biological Indicators or Bio-indicators are characterized preparations of a specific microorganism; it provides a defined and stable resistance to a specific sterilization. So basically, they are used to validate and confirm the integrity of sterilization processes. A widely recognized bio Indicator is a spore-forming bacteria.


The first form is adding spores to a carrier (Glasses, disks, filter paper, plastic, etc.) and packaged to maintain viability and integrity of the inoculated carrier.

- Carriers and primary packaging shall not contain any contamination (physical, chemical, or microbial) that would adversely affect the performance or the stability characteristics of the biological indicator.

- The carrier and primary packaging shall not be degraded by the specific sterilization process, which is used in a manner that will affect the performance of the biological indicator.

The second form is inoculating the spore into the representative product or a simulated one, if there is no practicality into putting it into the actual. The simulated product slightly differs from the actual product but acts as the actual product in test conditions or during actual production sterilization processing. The third form is the self-contained is intended for incubation after the sterilization process, which contains the growth medium for recovery of the process-exposed microorganisms. This form of bio-indicator and the self-contained growth medium can be considered a system. Self-contained biological indicators provide resistance to the sterilization process.

Three types of biological indicators

The selection of a biological indicator requires knowledge of the resistance of the biological indicator system to the specific sterilization process. It must be established that the biological indicator system provides a challenge to the sterilization process that exceeds the challenge of the natural microbial burden in or on the product.

Selection for Specific Sterilization Processes

Moist Heat— For moist heat sterilization process, spores of suitable strains of Bacillus stearothermophilus are commercially available as biological indicators and frequently employed. 9

Dry Heat— For dry heat sterilization, spores of Bacillus subtilis spp. are sometimes used to validate the process.

Ionizing Radiation— Spores of Bacillus pumilus have been used to monitor sterilization processes using ionizing radiation; however, this is a cedlining practice. Radiation dose-setting methods that do not use biological indicators have been widely used to establish radiation processes. Furthermore, certain bioburden microorganisms can exhibit greater resistance to radiation than Bacillus pumilus.

Selection for Specific Sterilization Processes (cont’d)

Bacillus stearothermophilus is the most prevalently used biological indicator for validating VPHP. Other microorganisms that may be useful as biological indicators in VPHP processes are spores of Bacillus subtilis and Clostridium sporogenes. Other microorganisms may be considered if their performance responses to VPHP are similar to those of the microorganisms cited above.

Selection for Specific Sterilization Processes (cont’d)

1. Wear complete lab attire and do the necessary precautions (e.g. washing of hands)2. Keep your workspace free of all unnecessary materials3. Disinfect work areas before and after use with 70% ethanol or fresh 10% bleach4. Replace caps on reagents, solution bottles, and bacterial cultures. 5. Inoculating loops and needles should be flame sterilized in a Bunsen burner before you lay them down.6. When you flame sterilize with alcohol, be sure that you do not have any papers under you.7. Treat all microorganisms as potential pathogens. Use appropriate care and do not take cultures out of the laboratory.8. Wear disposable gloves when working with potentially infectious microbes or samples9. Sterilize equipment and materials.10. Consider everything a biohazard. Do not pour anything down the sink. Autoclave liquids and broth cultures to sterilize them before discarding.11. Dispose of all solid waste material in a biohazard bag and autoclave it before discarding in the regular trash.12. Dispose of chemicals and other used materials to their appropriate containers or trash bins.

Microbiology Lab Practices and Safety Rules

Biological Safety Cabinet

- A biological safety cabinet (BSC) is used as a primary barrier against exposure to infectious biological agents. A BSC has High Efficiency Particulate Air (HEPA) filters. The airflow in a BSC is laminar, i.e. the air moves with uniform velocity in one direction along parallel flow lines. Depending on the design, a BSC may be vented to the outside or the air may be exhausted into the room. BSCs are not chemical fume hoods. A percentage of the air is recirculated in most types of Randall E. Hicks BSCs. HEPA filters only trap particulates, allowing any contaminant in non-particulate form to pass through the filter.

Laboratory Safety Equipment

1. Operate the cabinet for five minutes before and after performing any work in it in order to purge airborne contaminants.

2. Before and after use, wipe the surface of the BSC with a suitable disinfectant, e.g., 70%alcohol or a 10% bleach solution.

3. Place everything you will need inside the cabinet before beginning work, including a waste container. You should not have to penetrate the air barrier of the cabinet once work hasbegun.

4. Do not place anything on the air intake grills, as this will block the air supply.5. You should prevent unnecessary opening and closing of door because this will disrupt

the airflow of the cabinet.6. Always wear a lab coat while using the cabinet and conduct your work at least four

inches inside the cabinet.7. Place burners to the rear of the cabinet to reduce air turbulence.8. Do not work in the BSC while the ultraviolet light is on. Ultraviolet light can quickly injure

the eye.9. When finished with your work procedure, decontaminate the surfaces of any

equipment.10. Remove the equipment from the cabinet and decontaminate the work surface.11. Thoroughly wash your hands and arms.

Proper Use of BSCs:

- Biosafety levels are selected to provide the end-user with a description of the minimum containment required for handling different microorganisms safely in a laboratory setting and reduce or eliminate exposure to potentially hazardous agents. Containment refers to safe methods for managing infectious material in the laboratory environment. These biosafety levels are applicable to facilities such as diagnostic, research, clinical, teaching, and production facilities that are working at a laboratory scale.

Biosafety Levels and Practices

Examples of BSL1 Agents: Bacillus subtilus, Naegleria gruberi, many Escherichia coli, Infectious Canine Hepatitis Virus

BSL1 containment is suitable for work involving well-characterized agents not known to cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. A BSL1 lab requires no special design features beyond those suitable for a well-designed and functional laboratory. Biological safety cabinets (BSCs) are not required. Work may be done on an open bench top, and containment is achieved through the use of practices normally employed in a basic microbiology laboratory.

Biosafety Level 1 (BSL1)

Examples of BSL2 Agents: Bacillus anthracis, Bordetella pertussis, Brucella spp., Cryptococcus neoformans, Clostridium botulinum, Clostridium tetani, Helicobacter pylori, most Salmonella spp., Yersinia pestis, Mycobacterium leprae, Shigella spp., Human Immunodeficiency Virus, Human blood

The primary exposure hazards associated with organisms requiring BSL2 are through the ingestion, inoculation and mucous membrane route. Agents requiring BSL2 facilities are not generally transmitted by airborne routes, but care must be taken to avoid the generation of aerosols (aerosols can settle on bench tops and become an ingestion hazard through contamination of the hands) or splashes. Primary containment devices such as BSCs and centrifuges with sealed rotors or safety cups are to be used as well as appropriate personal protective equipment (i.e., gloves, laboratory coats, protective eyewear). As well, environmental contamination must be minimized by the use of hand washing sinks and decontamination facilities (autoclaves).


Examples of BSL3 Agents: Myobacterium tuberculosis, Salmonella typhi, Vesicular Stomatitis Virus, Yellow Fever Virus, Francisella tularensis, Coxiella burnetti

Laboratory personnel have specific training in handling these pathogenic and potentially lethal agents and are supervised by scientists who are experienced in working with these agents. These agents may be transmitted by the airborne route, often have a low infectious dose to produce effects and can cause serious or life-threatening disease. BSL3 emphasizes additional primary and secondary barriers to minimize the release of infectious organisms into the immediate laboratory and the environment. Additional features to prevent transmission of BSL3 organisms are appropriate respiratory protection, HEPA filtration of exhausted laboratory air and strictly controlled laboratory access.


Examples of BSL5 Agents: smallpox virus, Ebola virus, hemorrhagic fever viruses

This is the maximum containment available and is suitable for facilities manipulating agents that are dangerous/exotic agents, which post a risk of life threatening disease. These agents have the potential for aerosol transmission, often have a low infectious dose and produce very serious and often fatal disease; there is generally no treatment or vaccine available. This level of containment represents an isolated unit, functionally and, when necessary, structurally independent of other areas.

BSL4 emphasizes maximum containment of the infectious agent by complete sealing of the facility perimeter with confirmation by pressure decay testing; isolation of the researcher from the pathogen by his or her containment in a positive pressure suit or containment of the pathogen in a Class III BSC line; and decontamination of air and other effluents produced in the facility.


Whilst there are undoubtedly some who fear all microbes due to the association of some microbes with various human illnesses, many microbes are also responsible for numerous beneficial processes such as industrial fermentation (e.g. the production of alcohol, vinegar and dairy products), antibiotic production and as vehicles for cloning in more complex organisms such as plants. Scientists have also exploited their knowledge of microbes to produce biotechnologically important enzymes such as Taq polymerase, reporter genes for use in other genetic systems and novel molecular biology techniques such as the yeast two-hybrid system. Recent research has suggested that microorganisms could be useful in the treatment of cancer. Various strains of non-pathogenic clostridia can infiltrate and replicate within solid tumors. Clostridial vectors can be safely administered and their potential to deliver therapeutic proteins has been demonstrated in a variety of preclinical models.

Benefits of Microbiology

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Microbiology Report