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MICROORGANISMS

1. State the aims of classifying organisms

 Classification attempts to establish a universal  criteria for identifying organisms & assignment to groups (what belongs where) without creating confusion. This enables easier  study and understanding of  characteristics this organisms  

2.Describe the binomial nomenclature

 It is a system of classification where organisms are named by being given names of the two level taxa, genus and species, to name a specie. Binomial nomenclature includes:

Genus comes before species (e.g., Escherichia coli)

1. Genus name is always capitalized (e.g., Escherichia)

iii. Species name is never capitalized (e.g., coli)

1. Both names are always either italicized or underlined ( e.g Escherichia coli )
2. The genus name may be used alone, but not the species name (i.e saying or writing “Escherichia “ alone is legitimate while saying or writing “ coli” is not)
3. Name and describe the three ways in which colonies are classified in microbiology.

Colonies are defined as the visible mass of microorganisms originating from multiple cell divisions over a period of time. therefore, colonies are clones of the single mother cell. They cluster together as colonies on solid growth media on a petri plate or slats in a test tube. When microbes are grown on specific solid media like agar, they develop into a colony with morphological structures that are unique and can be used as the first step in identifying the possible group. A hand lens or magnifying glass is used to study the colony structure.

4. Draw a labelled generalized bacteria structure

5. Explain the function of the following bacteria structures
   1. Slime layers and capsules

This outermost surface of a bacterial cell that essentially forms a covering layer or a sort of envelope around the cell wall material is called slime layer if it is loose and slimy.  In some bacterial species, the material is compact and rigid, in which case it is called capsule. The slime layer/capsule provides protection to the organism against adverse environmental conditions. Thus, it protects the cell against desiccation, chemical antimicrobial agents, and antibiotics as well as phagocytosis by immune cells. In addition to its protective function, the extracellular polysaccharides (EPS) may invariably promote attachment of bacteria to each other and to the substratum in biofilms (for example, Streptococcus mutans). It can also function in virulence.

1. Cell wall

Found below the cell surface layer (slime layer/capsule) is the cell wall. The cell wall is one of the most important structures of the bacterial cell and one of the things which sets it apart from animal cells. The bacterial cell wall has two major roles to play: It protects the cell against osmotic rupture particularly in diluted media, and also against certain possible mechanical damage(s). Any breakdown in, or malformation of, the cell wall structure would lead to the loss of cytoplasmic contents and, consequently, death of the cell.

It is responsible for the rigidity and shape of the cell, their subsequent major division into Gram-positive and Gram-negative microorganisms, and their antigenic attributes.

Bacterial cell wall consists of a specific alternating arrangement of molecules of N-acetylglucosamine and N-acetylmuramic acid to form a glycan chain. A tetrapeptide string of amino acids attached to each molecule of N-acetylmuramic acid, while pentaglycine bridges interlink the tetrapeptide strings of adjacent glycan chain. The resulting polymeric structure is called peptidoglycan or muran. The peptidoglycan-based cell wall is unique to bacteria. It is absent in eukaryotic cells. It is the target of action of some antibiotics, which can selectively kill bacteria with little or no harm to the host.

1. Flagella

Flagella refer to thread-like structures attached to the surface of some bacteria. They enable bacteria to move and cells lacking flagella are nonmotile. Flagella are approximately 0.01 µm in diameter and 15 µm in length. They are made up of repeating units of a simple protein called flagellin.

The numbers of flagella per bacterial cell vary with bacterial species. Some have a single, polar flagellum and thus are described as monotrichous, whereas others are flagellate over their entire surface (peritrichous).

1. Fimbriae

Much like flagella, fimbriae consist of protein subunits. Fimbriae are, however, shorter (3 µm), finer, and more rigid than flagella. They are not involved in motility rather they serve as instruments of attachment of bacteria to surfaces and tissues, especially in the course of infection. Fimbriae are also responsible for haemagglutination and cell clumping in bacteria.

6. Give four differences between fungus and bacteria

• Most fungi are saprophytic in nature while bacteria are either autotrophic or heterotrophic
• Fungi have the cell made up of hyphae or mycelia while bacteria have their cells made up of cell walls
• Most fungi reproduce asexually by  spores , fragmentation , fusion and budding , while bacteria reproduce by binary fusion
• Most fungi are multicellular in nature while bacteria are purely unicellular

7. Using  a labeled diagram , illustrate the following types of  bacteria cells

 

8. Describe the culturing of anerobic bacteria

Anaerobic bacteria differ from aerobic bacteria in their oxygen requirement. Oxygen is toxic to anaerobes. Obligate anaerobes are bacteria that can live only in the absence of oxygen.  An anaerobic bacteria culture is a method used to grow anaerobes from a clinical specimen. Anaerobic bacterial cultures are performed to identify bacteria that grow only in the absence of oxygen and which may cause human infection. The methods of obtaining specimens for anaerobic culture and the culturing procedure are performed to ensure that the organisms are protected from oxygen. 

The keys to effective anaerobic bacteria cultures include collecting a contamination-free specimen and protecting it from oxygen exposure. Anaerobic bacteria cultures should be obtained from an appropriate site without the health care professional contaminating the sample with bacteria from the adjacent skin, mucus membrane, or tissue. Swabs should be avoided when collecting specimens for anaerobic culture because cotton fibers may be detrimental to anaerobes. Abscesses or fluids can be aspirated using a sterile syringe that is then tightly capped to prevent entry of air.

Tissue samples should be placed into a degassed bag and sealed, or into a gassed out screw top vial that may contain oxygen-free prereduced culture medium and tightly capped. The specimens should be plated as rapidly as possible onto culture media that has been prepared as prereduced anaerobically sterilized media (PRAS). Alternatively, media that contains a reducing agent such as dithiothreitol or palladium chloride and has been stored for one day in an anaerobic chamber can be used.

Broth and solid media should both be inoculated. The culture media should include anaerobic blood agar plates enriched with substances such as brain-heart infusion, yeast extract, amino acids, and vitamin K; a selective medium such as kanamycin-vancomycin (KV) blood agar or laked blood agar; and a broth such as brain heart infusion broth with thioglycolate or other reducing agent. The choice of media depends upon the type of specimen. Some commonly used media include prereduced peptone-yeast extract-glucose broth which is suitable for analysis of volatile products by gas chromatography; egg yolk agar for detection of lecithinase acitivity of Clostridium spp.; cycloserine-cefoxitin-fructose agar (CCFA) for isolation of Clostridium difficile from stool; and Bacteroides bile esculin agar for isolation of the Bacteroides fragilis group.

Cultures should be placed in an environment that is free of oxygen, at 95 °F (35 °C) for at least 48 hours before the plates are examined for growth. In addition, blood agar plates should be cultured both aerobically and in 10% carbon dioxide.

. An anaerobic glove box made of acrylic or plastic with sealed armholes and attached gloves is the most commonly used environment for anaerobic culture. The interior of the glove box or hood is supplied with a mixture of 10% hydrogen, 10% carbon dioxide, and 80% nitrogen from compressed gases.

Inoculation and transfers can be performed in the glove box without risk of oxygen exposure. The alternative to an anaerobic chamber is a jar such as the GasPak system. When using jars, several are needed to contain uninoculated media and subcultured plates (i.e., plates containing a colony transferred from the primary isolate). The GasPak system uses a disposable envelope containing citric acid, sodium bicarbonate, cobalt chloride, and sodium borohydride. When water is added these react to form hydrogen gas.

A catalyst, palladium, located in the lid converts the hydrogen and oxygen in the jar to water. Silica gel or paper towels are used to absorb the water, and an indicator strip of methylene blue is used to indicate anaerobic conditions. Methylene blue will be oxidized when the atmosphere contains oxygen causing formation of a blue color.

9. Distinguish between various types of culture media, give suitable examples in each case

Media can be classified in a number of ways i.e.  

• Solid, liquid or semisolid media. Solid media is referred to as  agar  while liquid media is referred to as broth
• Simple, complex ,synthetic or natural media
• Special purpose media – these include
• Basic media
• Enriched media
• Enrichment media
• Selective media
• Differential media
• Anaerobic and aerobic media
• Special purpose media. They include;

1. Basic media– these are simple media  that contain only  the necessary constituents  for growth e.g.  meat extract , peptones  and mineral salts . Examples are  peptone broth ,nutrient broth, and blood agar base .

Many microorganisms are capable of growing on basic media . They are also referred to as  general type media.

Basic media are often used in the lab to  prepare enriched media  and to maintain stock cultures , and for subculturing  of microorganisms  from differential or  selective media prior to accomplishing  serological and biochemical test .

Basic media are cheap ,

1. Enriched media– These are media enriched with whole blood , lysed blood ,serum, extra peptones  even amino acids so as to  support  the growth of pathogens  that require additional  nutrients or growth stimulants  e.g. sheep blood agar, chocolate blood agar ,horse blood agar

Sheep blood and horse blood is most preferred because they do not contain  inhibiting factors  . chocolate blood is made by heating  blood at 60^oc  which turns to chocolate brown color

1. Selective media– These are nutrient agar media  which have been added specific  chemicals  which will inhibitor prevent growth  of one group of bacteria  without inhibiting the other group e.g.  addition of crystal  violet  at specific concentration  will prevent  the growth of gram positive  without affecting growth of gram negative  bacteria

Selective media  are therefore used to grow special kind of bacteria  on a very unusual  organic compound  by adding a compound  which can stimulate  growth of one kind of bacteria  but inhibit the other  kind of bacteria

1. Differential media– These are nutrient agar media  in which certain  reagents or chemical have been incorporated  which will result in the kind  of growth change  in the bacteria present  which will permit the observer to  differentiate  between types of bacteria e.g.  if some bacteria are inoculated in blood agar media , some will be hemolysis (bust) while others will not . Therefore a certain  clear around a colony  is evidence of hemolysis .these can distinguish between  hemolytic and non-hemolytic bacteria. This media can be categorized into;

Assay media – these are media used for assay of vitamins, amino acids and antibiotics. They are also used for testing antibiotics

Enumeration media – these are media used for determining bacterial  population or number

Media for characterization – these are media used to determine the type of growth  produced by organisms  as well as their ability  to produce chemical changes

Maintenance media – they are media used to maintain viability  and physiological characteristic of culture

10. Describe the blood agglutination test

Blood agglutination  testing makes use of reactions between blood group antigens and antibodies—specifically the ability of antibodies to cause red blood cells to clump together when they bind to antigens on the cell surface, a phenomenon called agglutination. 

11. Describe how virus  reproduce

 Virus  reproduce through  a  replication cycle  which  can vary from virus to virus, there is a general pattern that can be described, consisting of five steps:

1. Attachment – the virion attaches to the correct host cell.
2. Penetration or Viral Entry– the virus or viral nucleic acid gains entrance into the cell.
3. Synthesis – the viral proteins and nucleic acid copies are manufactured by the cells’ machinery.
4. Assembly– viruses are produced from the viral components.
5. Release– newly formed virions are released from the cell.
6. Draw a labelled diagram of virus.

12. State four characteristics to consider in identification of protozoa in the laboratory

• They are single cell and microscopic
• They have flagella or cilia
• Their body shape is amoeboid

13. Distinguish between photoautotropism and chemoheterotropism

Photoautotrophism is the process of nutrition where organisms  utilize light to assimilate CO2. Chemoautotrophism  is where they  utilize chemical energy for assimilation of CO2. Chemoheterotrophism is where organisms which lack the ability to fix CO2 ,utilize already synthesized  organic compound as carbon source

14. Differentiate between gram positive and gram negative bacteria

Gram staining method was developed by Christian Gram.

Most bacteria are differentiated by their gram reaction due to differences in their cell wall structure.

Gram-positive bacteria are bacteria that stain purple with crystal violet after decolorizing with acetone-alcohol.

Gram-negative bacteria are bacteria that stain pink with the counter stain (safranin) after losing the primary stain (crystal violet) when treated with acetone-alcohol

Required reagents:

• . Gram’s Iodine
• . Acetone-Alcohol
• . Safranin

Procedure:

• Prepare the smear from the culture or from the specimen.
• Allow the smear to air-dry completely.
• Rapidly pass the slide (smear upper most) three times through the flame.
• Cover the fixed smear with crystal violet for 1 minute and wash with distilled water.
• Tip off the water and cover the smear with gram’s iodine for 1 minute.
• Wash off the iodine with clean water.
• Decolorize rapidly with acetone-alcohol for 30 seconds.
• Wash off the acetone-alcohol with clean water.
• Cover the smear with safranin for 1 minute.
• Wash off the stain wipe the back of the slide. Let the smear to air-dry.
• Examine the smear with oil immersion objective to look for bacteria.

Interpretation:

. Gram-positive bacterium ……………Purple

. Gram-negative bacterium …………..Pink

15. Draw a labelled diagram of Rhizopus.

16. Outline the economic importance of microorganisms
Microorganisms are useful to us in many ways. For example,

• Bacteria like Lactobacillus convert milk into curd.
• Bacteria are also involved in the making of cheese.
• Acetobacter aceti is used for producing acetic acid from alcohol.
• Yeast is used in the commercial production of alcohol, wine and bakery products.
• Some specific microorganisms are helpful in manufacturing of antibiotics.
• Microorganisms act as cleansing agents and decompose the waste products into manure.
• Dead or weakened microbes are used in the preparation of vaccines.
• Some bacteria fix atmospheric nitrogen and increase soil fertility.
• Algae, yeast, fungi or bacteria may be used as an ingredient or a substitute for protein-rich foods that are suitable for human or animal consumption.
• Some microorganisms are taken as probiotics, that are believed to provide health benefits when consumed.
  Microorganisms are harmful to us in many ways. For example, microorganisms, called pathogens cause disease in humans, plants and animals. Pathogens or germs enter a healthy body through air, water, contaminated food and infected person by direct or indirect contact or by the carrier. Common ailments like cold, influenza (flu), cough, polio, chicken pox are caused by viruses. Foot and mouth diseases in the cattle are also caused by viruses. Typhoid, tuberculosis (TB) are caused by bacteria. Anthrax a dangerous human and cattle diseases is also caused by bacteria.
• Diseases like dysentery and malaria are caused by protozoa. Ringworm is caused by fungi. Several microbes causes diseases in plants and thus reduces the yield. Citrus canker, a bacterial disease, affects trees of citrus fruit and is spread by air. Bhendi yellow vein mosaic disease is caused by a virus and is spread by insects in lady fingers. Rust of wheat is a fungal disease spread through air. Microorganisms that grow on our food sometimes produce toxic substances. These make the food poisonous causing serious illness and even death. This food-borne illness is called food poisoning

17. Define the term

• aseptic techniques

Aseptic technique means using practices and procedures to prevent contamination from pathogens. It involves applying the strictest rules to minimize the risk of infection. Healthcare workers use aseptic technique in surgery rooms, clinics, outpatient care centers, and other health care settings

• Innoculation

Innoculation is the introduction of a microorganism into a medium suitable for its growth..

18. Distinguish between bacteriocidal and bacteriostatic agent

Bactericidal products eliminate bacteria while bacteriostatic products keep bacterial populations in check by inhibiting replication.

A bactericidal substance is that these antimicrobial treatments directly kill bacteria. These agents “attack” microbes by affecting the cell wall, lipids, enzymes, or protein synthesis within the cell – sometimes even completing a combination of these mechanisms. By disrupting the cell wall structure of existing cells and inhibiting the formation of new cells, bactericidal substances cause bacterial cells to die off, therefore decreasing the amount found in the individual affected.

    Bacteriostatic treatments differ from bactericidal versions in that they inhibit the growth and multiplications of bacterial cells, rather than directly kill them.

Bacteriostatic agents can achieve this by obstructing the metabolic mechanisms of the bacterial cell, in most cases targeting the protein synthesis. While doing this does not cause outright cell death, it does effectively inhibit further growth and DNA replication of the bacterial cells.

When bacteriostatic agents are utilized, the treatment will regulate the number of bacterial cells. While the bacteria will not be eliminated, their numbers will not increase. Essentially, bacteriostatic treatments effectively keep bacterial cells from replicating.

Bacteriostatic substances produce reversible results. As mentioned in the introduction, very high treatment levels of bacteriostatic agents may eventually result in bactericidal characteristics – irreversible action and/or cell death.

19. List four methods of dry heat sterilization

Method	Principle/ Conditions	Advantages	Disadva	Uses
Dry Heat	• Thermal inactivation: destroys by oxidation	• Non-corrosive Simple design and principle	• Less effective than moist heat; • Requires longer times and/or higher temperatures	• Materials that are damaged by, or are impenetrable to, moist heat
Hot Air Oven	• 160-180oC for 2-4 hours	• Penetrates water-insoluble materials (e.g., grease and oil) • Less corrosive to metals and sharp instruments than steam	slow diffusion, penetration • Loading, packing critical to performance • Not suitable for reusable plastics	• Anhydrous materials, such as oils, greases and powders • Laboratory glassware, instruments • closed containers
Red-heat Flame	• Oxidation to ashes (burning)	• Rapid	• Initial contact with flame can produce a viable aerosol • Possibility of accidental fire	• Inoculating loops, needles
Incineration	• Oxidation to ashes (burning) • 1-60 minutes: temperature may exceed 1000?C	• Reduces volume of waste by up to 95%	• Improper use may lead to emission of pathogens in smoke • Require transport of infectious waste • Excess plastic (>20%) content reduces combustibility	• For decontamination of waste items prior to disposal in landfill

 

 

 

 

 

 

 

 

 

State the advantages of moist heat sterilization

Method	Principle/Conditions	Advantages	Disadva	Uses
Pasteurization	Heating to below boiling point (generally 77oC) for up to 30 minutes	• Can be used on heat sensitive liquids and medical devices Low cost	Not reliably sporicidal	• Milk and dairy products • some heat-sensitive medical equipment
Tyndallization ( Fractional S terilization)	Heating to 80-100oC for 30 mins on successive days, with incubation periods in between	Resistant spores germinate and are killed on the second and third days	Time consuming not reliably sporicidal	• Heat sensitive materials such as bacteriologic media, solutions of chemicals, biological materials
Autoclaving	Apply Steam under pressure  usually at  121oC/15 psi for               15 -90 mins (gravity displacement autoclave)  or at 132oC/27 psi for    4-20 minutes (pre-vacuum autoclave)	Minimal time required Most dependable sterilant for lab use	Loading and packing critical to performance Shielding dirt must first be removed Maintenance and quality control essential	• Penetration of sterile glassware, media and instruments • Decontamination of reusable supplies and equipment • Decontamination of infectious waste

 

20. State the limitation of ultra violent light (UV) as a sterilizing agent in the laboratory

UV  rays are  germicidal and can be used to reduce the number of pathogenic microorganisms on exposed surfaces and in air.

However, UV light has poor penetrating power; accumulations of dust, dirt, grease or clumps of microorganisms may shield microorganisms from the direct exposure required for destruction.

UV light presents skin and eye burn hazard, also factors such as lamp age and poor maintenance can reduce performance.

21. Distinguish between the two types of pasteurization

Pasteurization is the process of heating something up quickly then cooling it back down. Pasteurizing milk destroys 99.9% of disease-causing microorganisms and extends the shelf life to 16-21 days from the time it was packaged.

1. High Temperature Short Time

The most common method of pasteurization is High Temperature Short Time (HTST). This method involves using metal plates and hot water to raise the temperature of the milk to at least 161 °F (71 °C) for no less than 15 seconds, or 145 °F (62 °C) for 30 minutes, followed by rapid cooling.

1. Higher Heat Shorter Time

Similar to HTST pasteurization, Higher Heat Shorter Time (HHST) uses slightly different equipment and higher temperatures for a shorter time. Using HHST, milk can be heated anywhere from 191 °F (89 °C)  – 212 °F (100 °C) for its specified time (see chart below).

1. Ultra High Temperature

Another popular method of pasteurization is Ultra High Temperature (UHT). This process involves heating the milk using commercially sterile equipment and filling it under aseptic conditions into hermetically sealed packaging. The milk must be heated to 280 °F (138 °C) for at least two seconds, then rapidly cooling it down. UHT kills more bacteria (good and bad) and gives it a much longer shelf life. UHT milk does not need refrigeration, until opened, and is shelf stable for at least six months.

1. Ultra Pasteurized

Not to be confused with UHT, Ultra Pasteurized (UP) milk is heated using commercially sterile equipment, but it is not considered sterile because it is not hermetically sealed. Milk is heated to 280 °F (138 °C) for at least two seconds, then rapidly cooling it down. Since the milk is not hermetically sealed, it must be refrigerated with an average shelf life of 30 – 90 days.

22. Describe the phases of bacteria growth curve

Bacterial growth is the division of one bacterium into two daughter cells in a process called binary fission. bacterial growth in batch culture can be modeled with four different phases represented by the sigmoid curve below : lag phase (A), exponential or log phase (B), stationary phase (C), and death phase (D).

Bacterial growth curve: Bacterial growth in batch culture can be modeled with four different phases: lag phase (A), exponential or log phase (B), stationary phase (C), and death phase (D).

1. During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual bacteria are maturing and not yet able to divide. During the lag phase of the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs.
2. Exponential phase (sometimes called the log phase) is a period characterized by cell doubling. The number of new bacteria appearing per unit time is proportional to the present population. If growth is not limited, doubling will continue at a constant rate so both the number of cells and the rate of population increase doubles with each consecutive time period. For this type of exponential growth, plotting the natural logarithm of cell number against time produces a straight line. The slope of this line is the specific growth rate of the organism, which is a measure of the number of divisions per cell per unit time. The actual rate of this growth (i.e. the slope of the line in the figure) depends upon the growth conditions, which affect the frequency of cell division events and the probability of both daughter cells surviving. Exponential growth cannot continue indefinitely, however, because the medium is soon depleted of nutrients and enriched with wastes.
3. During stationary phase, the growth rate slows as a result of nutrient depletion and accumulation of toxic products. This phase is reached as the bacteria begin to exhaust the resources that are available to them. This phase is a constant value as the rate of bacterial growth is equal to the rate of bacterial death.
4. At death phase, bacteria run out of nutrients and die.

NB; Bacterial growth can be suppressed with bacteriostats, without necessarily killing the bacteria.

23. Explain factors to consider when selecting culture media

The selection culture media will depend on:

1. The major pathogens to be isolated, their growth requirements and the features by which they are recognized.
2. Whether the specimens being cultured are from sterile sites or from sites having normal microbial flora.
3. The cost, availability and stability of media.
4. The training and experience of laboratory staff in preparing, using and controlling culture media.

26. Explain the importance of transport media

Media containing ingredients to prevent the overgrowth of

commensals and ensure the survival of pathogenic bacteria when specimens cannot be cultured soon after collection. Examples include: Amies transport media, Stuart media and

Kelly-Blair media

27. Explain the steps involved in the preparation of culture media

The major processes during preparation of culture media

• Weighing and dissolving of culture media ingredients
• Sterilization and sterility testing
• Addition of heat-sensitive ingredients
• Dispensing of culture media
• pH testing of culture media
• Quality assurance of culture media
• Storage of culture media

1. Describe the preparation of nutrient agar , detailing their active ingredient and their use

• Suspend 28g of nutrient agar powder (CM0003B) in 1L of distilled water.
• Mix and dissolve them completely.
• Sterilize by autoclaving at 121°C for 15 minutes.
• Pour the liquid into the petri dish and wait for the medium to solidify.

29. Differentiate between defined and  complex media

Chemically defined media contain pure biochemical; hence, the chemically defined media contain exactly known chemical composition. On the other hand, complex media contain complex materials such as blood, milk, beef extract, yeast extract, etc., so the chemical composition of the complex media is unknown.

28. Explain the Aseptic technique during inoculation of culture media

• Decontaminate the workbench before and after the work of the day.
• Use facemask and gloves during handling highly infectious specimens.
• Flame sterilize wire loops, straight wires, and metal forceps before and after use.
• Flame the neck of specimen and culture bottles, and tubes after removing and before replacing caps and plugs.
  29. Outline  culturing bacteria  using deep culture technique

Deep culture is  solid medium made with agar and various nutrients and indicators. This type of culture is used for the growth of anaerobic bacteria which grow in the absence of oxygen and are inoculated by stabbing the media with a needle.

30.  Outline the working of an autoclave

An autoclave is a machine that uses steam under pressure to kill harmful bacteria, viruses, fungi, and spores on items that are placed inside a pressure vessel. The items are heated to an appropriate sterilization temperature for a given amount of time.It  works on the principle of moist heat sterilization. The high pressure inside the chamber increases the boiling point of water for the sterilization of equipment. The higher pressure also ensures the rapid penetration of heat into the deeper parts of equipment.

31. Outline sterilization  of glassware using a pressure cooker

 Make sure the items doesn’t fill the container by more than two-thirds. Put the lid on the jars and close tightly  otherwise it will explode. You can also cover jars and glassware with aluminium foil and keep it down with a piece of masking or autoclave tape

Fill the bottom of the pressure cooker with water to the depth of 1-2cm . Place a separator in the bottom of the pressure cooker so that the sample sits just on top of the water layer and is not directly touching the bottom of the cooker. In the video, we just used a spoon.

Seal the pressure cooker and place it on a hot plate at high heat. Make sure the pressure valve is completely open.

When steam begins to come out of the pressure valve opening, close it fully and turn the heat down to medium . When pressure have build up inside the pot, and steam leaves through the pressure valve you can start the timer. The required sterilization time is usually 20-30 min.

When the time is up, switch off the hot plate and remove the pressure cooker so it can cool down slowly for approx. 10-15min.

Then, gradually open the pressure valve until steam has stopped coming out.

Open the pressure cooker, close the lids of the samples. Careful, it will be hot.

32. Describe  sterilization by use of radiation

Radiation kills germs that can cause disease and neutralizes other harmful organisms. Sterilization with ionizing radiation inactivates microorganisms very efficiently and, when used for product wrapping, ensures that healthcare products are safe and can be relied upon.

33. Explain the sterilization of cleaned glass petri dishes in the laboratory

Standard protocol requires the use of autoclaves to sterilize petri dishes, as only high heat and pressure can effectively kill the full range of microorganisms, which can persist even under unusually harsh conditions.

34. Describe two types of pasteurization

            – flash

– holder

35. (a) Name any four physical sterillants

• Identify  the types of materials that can be sterilized  by each of the sterilant  above  
  36. State five characteristics  of an ideal microbiological culture media

It must contain all the ingredients required by the organism and in certain proportions. Basically there should be an energy source, various macro and micronutrients, vitamins etc. it must have a suitable pH. Moreover, it must be sterile so that the organism cultivated may form a pure culture.

37. Explain causes of resistance development in  microorganisms.

Resistance of bacteria to anti-microbial drugs

Production of enzymes that destroy or inactivate anti-microbials Eg. B.lactamase destroying B-lactam ring of penicillin

1. Altering permeability of bacterial cell membrane Eg. resistance to polymixin and tetracycline
2. Developing an altered structural target for the drug Eg. resistance to aminoglycosides and erythromycin
3. Developing an altered metabolic pathway that bypasses the

reaction inhibited by the drugs Eg. resistance to sulfonamides

4. Developing an altered enzyme that can still perform its metabolic function but is much less affected by the dru Eg. resistance to trimethop

37.  State reasons why samples for microbiological analysis may be rejected

4. Unlabelled or mislabelled samples
5. Leaky containers

A sample is unacceptable when the outside of the container is grossly    contaminated with the sample.

1. Contaminated samples– Contaminated samples are unacceptable.
2. Inappropriate sample sources- Samples that do not conform to the type of sample needed for the requested test(s) are unacceptable.  
3. Delayed transport time and sample processing

 

37. Giving one specific example in each case . classify culture media based on

• Nutrient factor
• Phase of growth

38. Explain the differential characteristics  of the following agar media

• Blood agar
• MacConkey agar

39. (a) Outline the preparation of bacteria lawn culture

Bacterial lawn is a term used by microbiologists to describe the appearance of bacterial colonies when all the individual colonies on a Petri dish agar plate merge to form a field or mat of bacteria. Bacterial lawns find use in screens for antibiotic resistance and bacteriophage titering.

Lawn cultures are prepared by flooding the surface of the plate with a liquid culture or suspension of the bacterium, pipetting off the excess inoculum, and incubating the plate. Alternatively, the surface of the plate may be inoculated by applying a swab soaked in the bacterial culture or suspension. After incubation, lawn culture provides a uniform growth of the bacterium.

40. List four uses of spread plate technique  in microbiology

It is used for evenly spreading cells to ensure growth of the isolated separate colonies. Further, it can be used for serial dilutions. The spread plate method is used for enrichment, enumeration and screening and selection of microorganisms. 

41. Outline the following culture techniques

• Plate count

The method measures the number of viable bacteria in a sample by exploiting the fact that when  an isolated cell is allowed to grow in  on a nutrient agar  it will give rise to one colony .

The number of colonies therefore found growing on a nutrient agar will tell  the number of  cells that were initially present in the  sample .there are two  different plating methods  i.e.  the pour plate method and the  spread plate method

Both methods first involve the dilution of samples in 10-fold increments. This is in order to disperse  the cells present in these samples  so that  the ideal number of cells in each sample are between 30- 300 cells or colonies .the dilution should be made from a sterile solution (or diluent)  which is usually  0.85 % NaCl in water (physiological saline ).

In pour plate method ,  about 0.1 – 1.0  of the final dilution is transferred  into a sterile petri dish  and then overlaid with melted nutrient agar hat have been cooled to 50^oc , the agar  is still liquid at these temperature.

The Petri dish is then gently swirled  to mix the bacteria with liquid agar  and is then left to harden . during hardening , the  individual cells are fixed  in place  and after incubation, they grow to form  distinguishable colonies

In spread plate  method ,  about  0.1 –0.2  ml of the final dilution is transferred into a plate originally containing  the already solidified nutrient agar  medium . The solution is then spread over the surface  of the agar with  a sterilized bent  glass rod .

After evenly spreading the sample on the solidified agar surface, the late is again  incubated  for a period of time  so as to allow the colonies to form  which can then be counted

By noting how much the sample was diluted and  by counting the number of colonies found to have grown in each dilution , a mathematical calculation can be done to  determine the number of cells likely to have been present in the original sample .

 It’s believed that each colony observed in the  plates came from one single cell  and therefore  cells clustered  together as one colony are referred to as  colony forming unit

b.Streak plate method 

It is a method which a sample of mixed bacteria is streaked several times along one edge of a Petri dish containing a medium such as nutrient agar. A loop is flamed and then touched to the first area to retrieve a sample of bacteria. This sample is then streaked several times in the second area of the medium. The loop is then reflamed, touched to the second area, and streaked once again in the third area. The process can be repeated in a fourth and fifth area if desired. During incubation, the bacteria will multiply rapidly and form colonies (Figure 1 ). 

Figure 1

Two processes for isolating bacteria from a mixed culture. (a) The streak plate technique. (b) The pour plate technique

41. State two ways in which protozoa are cultured in the laboratory

Protozoa  is cultured in shallow glass containers containing about 2 cm deep culture solutions e.g. chackleys media, which is prepared as a stock solution and diluted for use. It contains  

NaCl =16g

NaHCO3 = 0.8g

KCl =0.4g

NaHPO4=0.2g

NB: Never use deionized water because it contains phenol, which is harmful. In addition, extreme cleanness should be emphasized

For use, take 5cm3 of stock solution and dilute to 1dm3 with distilled water. Distribute the medium to culture dishes and about four boiled wheat grains in each dish and allows bacteria and molds to grow and finally colpedium, which will become food for Amoeba. Now inoculate with Amoeba

42. Name any two methods used to isolate pure culture of microorganisms

A pure culture theoretically contains a single bacterial species. There are a number of procedures available for the isolation of pure cultures from mixed populations. A pure culture may be isolated by the use of special media with specific chemical or physical agents that allow the enrichment or selection of one organism over another. The differential and selective procedures will be utilized later in this course.

Simpler methods for isolation of a pure culture include:

spread plating on solid agar medium with a glass spreader

streak plating with a loop.

The purpose of spread plating and streak plating is to isolate individual bacterial cells (colony-forming units) on a nutrient medium.

Both procedures (spread plating and streak plating) require understanding of the aseptic technique. Asepsis can be defined as the absence of infectious microorganisms. However, the term is usually applied to any technique designed  to keep unwanted microorganisms from contaminating sterile materials.

1. Spread Plate Technique

In this technique, the number of bacteria per unit volume of sample is reduced by serial dilution before the sample is spread on the surface of an agar plate.

1. Prepare serial dilutions of the broth culture as shown below. Be sure to mix the nutrient broth tubes before each serial transfer. Transfer 0.1 ml of the final three dilutions (10-5, 10-6, 10-7) to each of three nutrient agar plates, and label the plates.

2. Position the beaker of alcohol containing the glass spreader away from the flame. Remove the spreader and very carefully pass it over the flame just once (lab instructor will demonstrate). This will ignite the excess alcohol on the spreader and effectively sterilize it.
3. Spread the 0.1 ml inoculum evenly over the entire surface of one of the nutrient agar plates until the medium no longer appears moist. Return the spreader to the alcohol.
4. Repeat the flaming and spreading for each of the remaining two plates.
5. Invert the three plates and incubate at room temperature until the next lab period.
6. Streak Plate Technique

The streak plating technique isolates individual bacterial cells (colony-forming units) on the surface of an agar plate using a wire loop. The streaking patterns shown in the figure below result in continuous dilution of the inoculum to give well separated surface colonies. Once again, the idea is to obtain isolated colonies after incubation of the plate.

1. Label two nutrient agar plates No. 1 and No. 2.
2. Prepare two streak plates by following two of the 3 streaking patterns shown in the figure below. Use the 10-1 dilution as inoculum.
3. Invert the plates and incubate at room temperature until the next lab period.

 

43. Describe the various bacteria enumeration techniques

In the study of microbiology, there are numerous occasions when it is necessary to either estimate or determine the number of bacterial cells in a broth culture or liquid medium. Determination of cell numbers can be accomplished by a number of direct or indirect methods. The methods include standard plate counts, turbidimetric measurements, visual comparison of turbidity with a known standard, direct microscopic counts, cell mass determination, and measurement of cellular activity. In this exercise, you will compare three methods of bacterial enumeration: the standard plate count, turbidimetric measurement and direct microscopic counts.

1. Standard Plate Count (Viable Counts)

A viable cell is defined as a cell which is able to divide and form a population (or colony). A viable cell count is usually done by diluting the original sample, plating aliquots of the dilutions onto an appropriate culture medium, then incubating the plates under proper conditions so that colonies are formed. After incubation, the colonies are counted and, from a knowledge of the dilution used, the original number of viable cells can be calculated. For accurate determination of the total number of viable cells, it is critical that each colony comes from only one cell, so chains and clumps of cells must be broken apart.

However, since one is never sure that all such groups have been broken apart, the total number of viable cells is usually reported as colony-forming units (CFUs) rather than cell numbers. This method of enumeration is relatively easy to perform and is much more sensitive than turbidimetric measurement. A major disadvantage, however, is the time necessary for dilutions, platings and incubations, as well as the time needed for media preparation.

1. Turbidimetric Measurement

A quick and efficient method of estimating the number of bacteria in a liquid medium is to measure the turbidity or cloudiness of a culture and translate this measurement into cell numbers. This method of enumeration is fast and is usually preferred when a large number of cultures are to be counted.

1. Although measuring turbidity is much faster than the standard plate count, the measurements must be correlated initially with cell number. This is achieved by determining the turbidity of different concentrations of a given species of microorganism in a particular medium and then utilizing the standard plate count to determine the number of viable organisms per milliliter of sample. A standard curve can then be drawn (e.g., this lab protocol section), in which a specific turbidity or optical density reading is matched to a specific number of viable organisms. Subsequently, only turbidity needs to be measured. The number of viable organisms may be read directly from the standard curve, without necessitating time-consuming standard counts.

Turbidity can be measured by an instrument such as a colorimeter or spectrophotometer. These instruments contain a light source and a light detector (photocell) separated by the sample compartment. Turbid solutions such as cell cultures interfere with light passage through the sample, so that less light hits the photocell than would if the cells were not there. Turbidimetric methods can be used as long as each individual cell blocks or intercepts light; as soon as the mass of cells becomes so large that some cells effectively shield other cells from the light, the measurement is no longer accurate.

Before turbidimetric measurements can be made, the spectrophotometer must be adjusted to 100% transmittance (0% absorbance). This is done using a sample of uninoculated medium. Percent transmittance of various dilutions of the bacterial culture is then measured and the values converted to optical density, based on the formula: Absorbance (O.D.) = 2 – log % Transmittance. A wavelength of 420 nm is used when the solution is clear, 540 nm when the solution is light yellow, and 600-625 nm is used for yellow to brown solutions.

1. Direct Microscopic Count

Petroff-Hausser counting chambers can be used as a direct method to determine the number of bacterial cells in a culture or liquid medium. In this procedure, the number of cells in a given volume of culture liquid is counted directly in 10-20 microscope fields. The average number of cells per field is calculated and the number of bacterial cells ml-1 of original sample can then be computed. A major advantage of direct counts is the speed at which results are obtained. However, since it is often not possible to distinguish living from dead cells, the direct microscopic count method is not very useful for determining the number of viable cells in a culture.