MICRO LAB FINAL
Give examples of inappropriate use of antibiotics that can cause superbugs.
-not waiting to see of bacteria is resistant to antibiotic prior to prescribing, treating for viruses, treating just because -Over-use and misuse of antibiotics -Not taking for the prescribed duration -asking for it when you have a viral infection -exacerbates the development of drug-resistant bacteria, often called superbugs
Define simple stain
Use of a single dye to color a bacterial cell reveals shape, size, and arrangement
What color are acid-fast positive organisms
pink/red
What is required for transduction
transfer of DNA from one bacterium to another, using a VIRUS as a vector bacterial DNA is transferred from a door cell to a recipient cell inside a virus that infects bacteria called a bacteriophage.
Know the 5 antibiotics from the in the powerpoint, how they work, and what they work against
amoxicillin clavulinic acid: (A/AMC) -inhibits the synthesis of bacterial cell walls -*gram negative bacteria are not generally susceptible* -works well with Gram + bacteria bc of thick peptidoglycan (nag&nam) gentamicin(GM) / tetracycline (TE) -binds the 30S subunit of the bacterial ribosome, interrupting protein synthesis -effective against both gram - & + bacteria erythromycin(E) / chloramphenicol (C) -binds to 50S ribosomal subunit preventing peptide bond formation interrupting protein synthesis -prevent amino acids from being joined together
What are nosocomial infections? What % of these are resistant to at least one antibiotic?
an infection whose development is favoured by a hospital environment infections acquired in the hospital Studies show that ~70% of bacteria that cause nosocomial infections are resistant to at least one antibiotic commonly used to treat them
Why is it important to test for antibiotic sensitivitity
because if an organism is identified in a patient sample, we must figure out what type of antibiotic it should be treated with.
What color are acid-fast negative organisms
blue/green
Describe the role of macrophages in immunity
can play a response initiating role as antigen presenters Foreign substances in lymph that enter a lymph node may be phagocytized by macrophages Following phagocytosis of foreign material, macrophages process the antigen for use by the lymphocytes, thus initiating an immune response. Macrophages also stimulate proliferation of lymphocytes.
1. A student makes a smear by applying a bacteria onto the slide. A simple stain is performed, but when the student looks under the microscope, no organisms are seen. What did the student do wrong when making the smear?
forgot to heat fix - more then likely washed away the bacteria during the making process of the smear. - bacteria must be firmly attached during the washing and staining.
Are acid-fast organisms Gram positive or Gram negative?
gram-positive
how might gram + and gram - react with the agar (macconkey) know how bacteria react w the different agars
if we have a gram + that is lactose fermenting =you would see pink agar with no colonies
Measure and interpret the results of one antibiotic disk
measure in mm measure across
What type of agar is used with the Kirby-Bauer test
muller - hinton agar (MH) •Non-selective, non-differential =supportive agar •allows for Broad growth of a variety of bacteria •Contains starch •Absorbs toxins released from bacteria that may interfere with the antibiotics •Loose agar •Allows for better diffusion of the antibiotics
1. Describe the structure of a Gram negative cell wall.
negative cell envelope is peptidoglycan. Single layer of peptidoglycan (much thinner on whole), surrounded by a plasma membrane and an extra outer membrane composed of phospholipids, lipopolysaccharides, and lipoprotein.
What color are Gram negative organisms?
pink
Describe the structure of a Gram positive cell wall
plasma membrane and a thick layer of peptidoglycan
MacConkey Agar
selective and differential media selects for gram (-) bacteria inhibits the growth of (+) bacteria -selective for a specific type of bacteria (only gram -) -differentiates between gram (-) bacteria that metabolize lactose and those that do not
What can a simple stain determine
size shape arrangement of cells
Describe biochemical tests that can be performed on bacteria.
the most definitive way to identify bacterial species.- Each biochemical test helps determine a property or characteristic specific to a certain bacterial species. These tests determine which growth media the bacteria will grow on and identify the end products of their metabolic processes, such as the wastes they excrete.
What do basic dyes stain? Why
used to stain bacteria cells - because most bacteria is negatively charged, so -positively charged basic dyes stick to bacteria walls. include crystal violet, methylene blue, safranin, carbolfuchsin
What do acidic dyes stain? Why
used to stain the background (cells have a negative charge) In acidic (anionic) dyes, negative part is colored. Example: eosin (which binds to positively charged molecules, such as some amino acids.) Positively charged, cationic chromophores are called basic dyes because they combine with and stain acidic structures; further, they work best under basic (higher pH) conditions. -acid dyes are negatively charged (an ionic -)
What is this method called? the Kirby Bauer method used to determine the sensitivity or resistance of a bacterium to an antimicrobial
used to test a panel of antibiotics to see which one is most effective to treat an organism we tested PS.Aeruginosa (gram-) and S. Aureus (gram +) •An antibiotic disk is placed on the agar and the antibiotic diffuses out, creating a concentration gradient
Know how to measure a zone of inhibition (in millimeters).
usually 20-25mm 1st-measure diameter of the zone of inhibition (all the way across the zone) -measure in mm/the small little notches
What does it mean to say a bacterium is resistant to an antibiotic AKA-Antibiotic resistance
when germs like bacteria and fungi develop the ability to defeat the drugs designed to kill them. That means the germs are not killed and continue to grow. Infections caused by antibiotic-resistant germs are difficult, and sometimes impossible, to treat.
Name 2 superbugs (antibiotic resistant bacteria) and how they are transmitted
•4 Superbugs (There are many more out there) •MRSA - methicillin/oxacillin-resistant Staphylococcus aureus. •VRE - vancomycin-resistant enterococci. •ESBLs - extended-spectrum beta-lactamases. •PRSP - penicillin-resistant Streptococcus pneumonia. •+ CREs - carbapenem-resistant Enterobacteriaceae.
Describe the procedure for making a smear from a liquid culture -broth in a test tube
•: no additional liquid is needed •Use a sterile loop, -place a couple loop fulls of broth onto the slide - sterilize loop between touching the slide and dipping your loop
1. Describe the procedure for making a smear from an agar plate (bacterial culture growing on a solid agar plate).
•Add one drop of water to your slide •Use sterile inoculating needle to pick one colony- just a slight touch needed •Mix bacteria into water onto slide and try to spread out as thin as possible
What is PRSP resistant to and how is it transmitted? penicillin-resistant Streptococcus pneumonia.
•Can cause bacterial pneumonia and meningitis. •Also can cause bloodstream infections and ear and sinus infections.
What is VRE resistant to and how is it transmitted? vancomycin-resistant enterococci.
•Enterococci resistant to the antibiotic vancomycin. •Plasmid-mediated. •Can cause serious infections. Enterococci strains can live naturally in our intestines and on our skin.
Tips
•Keep the slide clean, only hold the slide on the sides. You may want to clean your slide with ethanol first. •Know when to sterilize! •Try to make the smear nice and thin. •Air dry to reduce cell shrinkage BEFORE heat fixing. •Don't overcook when heat fixing. •Weak adherence - don't wipe off your sample. •I would suggest using at least two different dyes.
•Extended-spectrum beta-lactamase (ESBL)-producing Gram Negative Bacteria
•Plasmid-mediatedà----easily transfer DNA code for ESBL enzymes. •Beta-lactamase: enzyme produced by some bacteria that can break down the beta-lactam ring in some antibiotics. •Predominately Klebsiella pneumoniae and E. coli, but found throughout Enterobacteriaceae.
3 Main Reagents for Acid Fast Staining
•Primary Stain: Carbolfuchsin - mixed with phenol (necessary so the carbolfuchsin can penetrate the mycolic acid) All cells will be stained pink. •Decolorizer: Acid Alcohol - 95% alcohol and hydrochloric acid; Decolorizes Non-AFB: colorless; Carbolfuchsin remains in AFB cells: remain pink. •Counterstain: Methylene Blue - stains non-AFB cells blue.
•Methicillin-Resistant Staphylococcus aureus (MRSA)
•Resistant to Methicillin and related beta-lactam antibiotics. •Patients who undergo invasive medical procedures or have weakened immune systems are at risk for Hospital-Associated MRSA (HA-MRSA). •1981 Community-Associated MRSA (CA-MRSA) •Identified in populations that share close quarters or more skin-to-skin contact - military, prisons, day cares.
•Carbapenem-resistant Enterobacteriaceae (CREs)
•phenotypic definition and it includes bacteria that are not susceptible to carbapenems via more than one type of mechanism. •Resistant to many available antibiotics. •One report cites CREs can contribute to death in up to 50% of patients who become infected.
Why is it desirable to combine S. aureus with acid-fast organism when using an acid-fast staining technique?
-where our control comes in -staphylococcus aureus is a negative control -we can use to make sure our stain is done properly
Give an example of a dye we used to perform a simple stain
***methylene blue*** safranin crystal violet carbolfuchsin
Difference between sterilization and decontamination
*Sterilization is a type of decontamination but not all decontamination techniques result in true sterilization* Decontamination is a cleaning process that decreases antimicrobial elements on surfaces. Types of decontamination are disinfection, antisepsis, and sterilization. General decontamination kills some bacteria and fungi while deactivating viruses. Sterilization kills all microorganisms, viruses, and bacterial spores.
For the diagnosis of what diseases is the acid-fast stain performed
+ stain diagnostic for TB (Tuberculosis) Leprosy
Explain the role of the bone marrow and thymus in immunity
-The thymus produces hormones that regulate T-cell maturation and serves as the incubator against infections. -primary lymphatic organ -It forms part of the immune system -T lymphocytes mature -bone marrow has hematopoietic stem cells -produces RBC -leukocytes fully develop here (that prevent infection) -T& b cells originate here -b cells mature
preparing a smear from a solid media -plate
-add one drop of water to your slide -use a sterile inoculating needle to pick one colony-just a slight touch is needed -mix bacteria into water onto slide and try to spread out as thin as possible
What CAN"T the Gram stain tell you about a bacteria
-does not identify individual species within a group -cant tell you the difference between cells w the my-colic acid-this is the acid fast stain Still, the Gram stain is widely used as it allows an experienced microbiologist to detect the presence of bacteria, and decide if a bacteria is Gram-positive or Gram-negative, Knowing the Gram type, and seeing the morphological shape of a bacteria aid in selecting the initial antibiotic needed to treat a patient.
after the smear is done for simple stain
-flood the slide with stain -let sit for usually 1 min -rinse stain -blot with bibulous paper -view your finished slide (100x) ---oil will not hurt your prepared slide,as long as you blot when finished VS. wiping oil from the slide for storage
Why are basic stains used more successfully on bacteria than acidic dyes
-increases contrast-makes cells more visible **because most cells are negatively charged** -so the basic dyes (+) are attracted to the cell walls (-) basic dyes - have positive charges and - the bacterial cell walls are negative, so they attract -cationic + (chromophore) -stains cells "positive staining" Acid dyes -are negatively charged, so - the negative cell walls are not attracted to it -anionic - (chromophore) -stains background "negative staining"
What are the criteria for a chemical compound to be considered an antibiotic
1. chemical substance produced by a microbe (bacteria or fungi) 2. stops the growth or kills other microorganisms 3. effective in small doses •If an organism is identified in a patient sample, (urine/sputum/blood) we must figure out what type of antibiotic it should be treated with •Some antibiotics can be broad spectrum (effective against both G+ & G-) or they can be specific against a certain type of organism
3 goals to a good smear: -how a smear is prepared will determine how well your stain will come out
1.Prepare a thin smear •thick smears make it harder to see individual cells and their arrangement, gives false staining results -use a little bit of bacteria 2.Insure that shrinkage of cells does NOT occur = Air Dry before Heat •IF YOU DONT=This distorts the cells, give improper representation of the cells shape/size -to ensure the cells remain in the shape that they actually are 3.Making sure the cells adhere to the slide = heat fix •If you forget to heat fix, your cells will wash right off
1. Describe the step by step procedure for performing a gram stain. Assume smear preparation and heat fixing has already been done. Be sure to name the dyes used.
1: prepare the smear 2: apply the primary stain (flood the smear with crystal violet) (the dye is positively charged and is attracted to the negative charge of a bacteria cell wall) -allow to stand for 1 min, rinse with water and removes excess stain-all the cells are purple 3: apply the mordant (flood the smear with iodine) (the iodine forms an insoluble complex with the crystal violet to anchor it to the cell wall) -allow to stand for 1 min 4: Rinse with water and remove excess iodine 5: (have water ready) you will drip decolorizer (80% methanol + 20% acetone) across the slide about 2-3 seconds (doing this removes the outer membrane of the gram negative bacteria) 6: rinse! Rinse immediately with water to remove excess alcohol -Gram + cells are purple/G - are colorless 7: counter stain; flood the slide with Safranin solution and let stand for 1 min -so gram - will pick up that dye 8: Rinse (with water to remove excess stain), dry (blot dry), and observe (under oil immersion) -were left with purple G+ cocci/pink G- bacillus
Describe the step-by-step procedure for performing an acid-fast stain. Be sure to name the dyes used.
1:Prepare the smear -staphylococcus aureus is a negative control for the stain (make sure the stain was done correctly) and mycobacterium smegmatis which is acid fast + 2:Add the primary stain Carbolfuchsin -the dye is positively charged and is attracted to the negative charge of the bacterial cell wall -allow to stand for 5 min, rinse w water to remove excess stain 5:Flood the slide w decolorizer (ACID-alcohol) across the slide about 30 sec -this removes the peptidoglycan layers of the gram positive and negative bacteria -but the mycolic acid of the acid fast positive bacteria remains (the acid fast + cells retain the carbolfuchsin dye) 7:Counterstain -flood the slide with methylene blue solution -let stand for 30 seconds -rinse w H2O -blot w bibulous paper -the (-) cells stain blue (cocci) / (+) stain pink (rods) -look at it under the microscope at 100x/oil immersion
Which stains are used to differentiate between the cell walls of bacteria
A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria. Conversely, the the outer membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-iodine complex and the color is lost. A counterstain, such as the weakly water soluble safranin, is added to the sample, staining it red. Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram positive cells. However, the decolorized Gram negative cells are stained red.
What is the difference between a simple stain and a differential stain
A simple stain determines size, shape, and arrangement of cells but cannot differentiate between types of bacteria. - uses one dye A differential stain uses 2 or more dyes to differentiate between organisms or between cell structures.
gram stain
A staining method that distinguishes between two different kinds of bacterial cell walls.
Define differential stain
A type of stain that can be used to distinguish between different types of bacteria B/c differentiates between Gram + and Gram - A stain that uses more then one color dye
Compare and contrast innate and adaptive immunity INNATE: nonspecific The part of your immune system that you are born with. It is not specific to any pathogen (antigen) and thus, not improved by repeated exposure (no memory). The body's first line of defense; holds pathogens "at bay" until the adaptive immune system can ramp up.• Physical components: skin, endothelium & epithelium, coughing, sneezing, crying, urination, and normal flora • Biochemical components: complement, lysozyme, pH, interferons • Cells: NK cells & phagocytes (neutrophils, monocytes, macrophages, dendritic cells)
ADAPTIVE:specific Immunity that is learned through exposure to a foreign substance or pathogen (antigen). It is specific for the particular antigen and is enhanced by repeated exposure, thus it improves over time and has immunological memory. • No physical components • Biochemical components: antibodies & cytokines • Cells: B&T lymphocytes and dendritic cells
Read the stain (Positive/negative, color and shape of each type of bacteria)
Acid fast bacteria remain red while nonacid fast bacteria stain in blue At the end of the gram stain, gram negative bacteria will be observed in pink colour while grams positive bacteria will be observed in purple colour.
What is the morphological difference between acid-fast organisms and non-acid-fast organisms (what chemical is found in the cell wall of acid-fast organisms?
Acid fast stain (or carbolfuchsin) binds only to bacteria that have a waxy cell wall. This cell wall contains a hydrophobic waxy lipid known as **mycolic acid** which occupies 60% of the cell wall. Due to the hydrophobic property, water soluble materials are prevented from entering the cytoplasm. That is why this bacteria is unable to be stained by water soluble dyes such as methylene blue. Carbolfuchsin is composed of phenol and fuchsin so that it can be penetrated up to the cytoplasm. During the acid alcohol decolorization step, the acid alcohol is prevented entering the cytoplasm due to the presence of hydrophobic mycolic acid, thus it is unable to remove carbolfuchsin from the bacteria cell. So the primary dye will remain in the cytoplasm even after the decolorization step.
What does the zone of inhibition vary with?
An area around the wafer where the bacteria have not grown enough to be visible •the diffusibility of the agent •the size of the inoculum •type of medium
Describe what antibodies are, what roles they play, and how they work
Antibodies are specialized Y-shaped proteins made by the immune system. They help the body fight disease by "recognizing" viruses, bacteria, and other pathogens by the molecules on their surface called antigens -can bind to a pathogenic antigens -can work with receptors on diff types of cells -helps pathogens from interacting with our cells in our body -cant perform phagocytosis of pathogens bc thats what macrophages do
Describe the roles of B cells and T cells in adaptive immunity.
B CELLS -They are mainly involved with antibody production. They can develop into plasma cells, which produce the most antibodies. -They can develop into either plasma or memory cells, and are made in the bone marrow. -The can present antigens to T cells. -responsible for humoral immunity that is mediated by circulating antibodies -self reactive (very important) -would not self react w antibodies that could enhance the speed of wound healing by recognizing damaged cells and helping to remove them T CELLS -responsible for cell mediated immunity -T cells kill targets directly or stimulate the activity of other leukocytes -react to intracellular pathogens/cancer cells
What type of paper do we use to dry the slide
Bulbous paper (blot with this paper)
GENUS - Taenia pisiformis
dog/ rabbit tapeworm PHYLUM PLATYHELMINTHS
Labster Virtual Labs:
Control of Microbial Growth
What type of bacteria are the "nightmare" superbugs (Gram positive or Gram negative)? Why are they this type?
Gram negative It is more armored than gram positive bacteria -gram-negative bacteria have a unique outer membrane. -This outer membrane excludes certain drugs and antibiotics from penetrating the cell
Be able to differentiate between a Gram stain and an acid fast stain under the microscope. The color The color of a gram stain is either going to be purple or pink The color of an acid fast stain is Hot pink and Blue
Gram negative bacteria are observed in pink colour gram positive bacteria are observed in purple colour. Acid Fast bacteria are observed in red colour non acid fast bacteria are observed in blue colour.
1. A student makes a smear by applying a bacteria onto the slide. A simple stain is performed, but when the student looks under the microscope, the organisms are misshapen and look weird. What did the student do wrong when making the smear?
Forgot to air dry before heat fix upon washing the student may have washed too much leading to Discoloration and making them look Misshaped
What morphological difference does the Gram stain distinguish bacteria based on -chemical properties of their cell walls -physical properties of their cell walls
Gram +Bacteria that stain purple. 80%-90% of the gram+ cell wall is peptidoglycan, much thicker layer. Gram - Bacteria that stain pink 10%-20% of gram-negative cell envelope is peptidoglycan. Single layer of peptidoglycan (much thinner on whole), surrounded by an outer membrane composed of phospholipids, lipopolysaccharides, and lipoprotein.
1. Explain how the gram stain works. Do NOT just repeat the procedure. Tell me why it works the way it does. -can differentiate the susceptibility of antimicrobial drugs to pick the right one -morphology-shape and structure of the cell wall Being a mordant, gram's iodine forms a complex with crystal violet in the stain that has attached more tightly to the cell wall of gram positive bacteria than that of the gram negative bacteria. Whereas the gram positive bacteria stain violet as a result of the presence of a thick peptidoglycan layer in the walls of their cell, the gram negative bacteria stain pink, due to the thinner peptidoglycan layer in their cell wall (a thicker peptidoglycan layer allows for the retention of the stain, but a thinner layer does not).
Gram-positive organisms: -Have a thick peptideoglycan cell wall that traps the crystal violet/iodine crystals -Have no outer membrane Gram-negative organisms: -Have a relatively thin peptidoglycan cell wall -Have an outer layer membrane that gets stripped with alcohol wash. -Stripping away outer membrane allows crystal violet/iodine crystals to be easily released
Describe transduction The process of using a bacteriophage to move pieces of chromosomal DNA from one bacterial cell to another -where we have the virus that takes the plasmid from one bacteria and goes over to some other location to another bacteria and gives that resistance gene to the other type of bacteria -bacteria does not need not be in the same place -need a virus -need a resistant gene in one bacteria
Horizontal gene transfer(HGT) is the movement of genetic elements between cells that are not direct progeny. HGT allows gene transfer between different bacteria species. Horizontal gene transfer may happen in the following ways: ● Conjugation ● Transformation ● Transduction
Define "zone of inhibition"
If the antibiotic inhibits or kills the test organisms, there will be a zone of no growth around the disk The zone where bacteria can't grow around a given antibiotic.
Describe how to read a Kirby-Bauer plate
If the organism is killed or inhibited by the concentration of the antibiotic, there will be NO growth in the immediate area around the disc: This is called the zone of inhibition Place the metric ruler across the zone of inhibition, at the widest diameter, and measure from one edge of the zone to the other edge. HOLDING THE PLATE UP TO THE LIGHT MIGHT HELP. Use millimeter measurements. The disc diameter will actually be part of that number. If there is NO zone at all, report it as 0---even though the disc itself is around 7 mm. Zone diameter is reported in millimeters, looked up on the chart, and result reported as sensitive, resistant, or intermediate. Record the results for your table, as well as other tables, in the table.
What is the first test you should perform when identifying an unknown bacteria specimen
Interpret a Gram stain, then culture your sample using differential media and perform the catalase, oxidase and indole biochemical tests on it. Critically combine the results of these assays in order to identify the unknown bacterium
Describe how the different methods of sterilization and decontamination work ionization filtration Decontamination is a process or treatment that renders a device, instrument, or work surface safe to handle. Wet Heat (steam)-steam sterilization using an autoclave is the gold standard method of inactivating(denatures) all organic pathogens including spores and prion proteins. -w/o proteins nothing can replicate or remain infectious Dry Heat-Baking objects in an oven is an alternative to wet heat sterilization where an object may be intolerant of moist conditions (for example due to corrosion). Chemical sterilants react with proteins and membranes to destroy organic pathogens Ethylene oxide chambers use toxic ethylene oxide gas to kill organic pathogens.
Liquid sterilants like peracetic acid and glutaraldehyde require objects to be completely immersed for a verified period of time to achieve sterilization. Both forms of sterilization by irradiation inactivate microorganisms by damaging their DNA either directly or indirectly. Non-ionizing radiation with ultraviolet (UV) light is a lower energy process than high energy ionizing radiation techniques using gamma rays or X-rays. As such non-ionizing UV radiation can't penetrate objects and can only be used to sterilize surfaces.
Describe how MacConkey agar works? How is it selective and differential
MacConkey agar is an indicator, a selective and differential culture medium for bacteria designed to selectively isolate Gram-negative and enteric bacilli and differentiate them based on lactose fermentation. The crystal violet and bile salts inhibit the growth of Gram-positive organisms which allows for the selection and isolation of gram-negative bacteria. Enteric bacteria that have the ability to ferment lactose can be detected using the carbohydrate lactose, and the pH indicator neutral red.
a. What is the mordant? What does the mordant do?
Mordant= IODINE Binds to crystal violet and intensifies stain -keeps the crystal violet in the membrane of the cell walls of gram + bacteria It is being added to a stain to give color to different kinds of organisms. With the use of mordant in microbiology, organisms are thoroughly and accurately identified. The definition of mordant is a chemical that keeps the dye in place
Ornithine Decarboxylase Test Test to determine if the microbe can use the amino acid ornithine as a food source. Lysine Decarboxylase Test Test to determine if the microbe can use the amino acid lysine as a food source. Citrate Utilization Test -Test to determine if the microbe can use citrate as its sole source of food. It is often used to differentiate between members of Enterobacteriaceae.=CO2 is produced -will turn from green to blue Voges-Proskauer Test (VP Test) -Test to determine if the microbe produces acetoin as a fermentation product from glucose.=will turn from brown to red to pink
Oxidase -Test to identify microorganisms containing the enzyme cytochrome oxidase (important in the electron transport chain). H2S -Test to determine if the microbe reduces sulfur-containing compounds to sulfide during metabolism. Glucose Fermentation Test -Test to determine if the microbe can ferment the sugar glucose.=PH indicator turns yellow Lactose Fermentation Test -Test to determine if the microbe can ferment the sugar lactose as a food source.=turn from red to yellow
Be able to differentiate between a Gram stain and an acid fast stain under the microscope.
Read the stain (Positive/negative, color and shape of each type of bacteria) GRAM STAIN purple = (+) pink= (-) ACID FAST pink= (+) blue= (-) RELATED -both differential -both help to visualize structures in the membrane
Which bacterium is more *sensitive* to antibiotic, Pseudomonas aeruginosa or Staphylococcus aureus? Why? An antibiotic sensitivity test can help find out which antibiotic will be most effective in treating your infection Antimicrobial resistance happens when germs like bacteria and fungi develop the ability to defeat the drugs designed to kill them. That means the germs are not killed and continue to grow. Resistant infections can be difficult, and sometimes impossible, to treat
Staphylococcus Aureus bc it is G (+) EX: for pseudomonas aeruginosa (G-) if it is resistant to the antibiotic the zone of inhibition will be less than< 13mm ---------(growing) if it is sensitive to the antibiotic the zone of inhibition will be greater than >17 mm (dying) EX:staph aureus <13mm= resistant >17mm=sensitive ---------
Decontamination reduce pathogens to levels considered safe to handle -kills some bacteria and fungi while deactivating viruses -broad term
Sterilization The process that completely destroys all microbial life, including spores. -kills everything -more specified decontamination
Compare and contrast B cells and T cells T cells and B cells are central to the human immune system. B cells and T cells operate in the adaptive immune response--the immune system's third and final line of defense. T cells work with B cells in their distinct roles in the immune system. B cells mature in the bone marrow. Once activated, B cells have binding sites that are specific to a pathogen. When the antigen is present, it binds to the receptor on the B cell. This triggers the B cell to grow and clone itself. The clones become either plasma cells or memory cells. The plasma cells generate massive amounts of antibodies and release them into the body. The antibody binds to the antigen signaling the cells of the internal defenses to come and kill the pathogen. The plasma cells die within a few days.Memory cells do not secrete antibodies. Instead, memory cells retain the antibody so that it can be used anytime the antigen is re-encountered. Memory cells are important because they help the body mount a faster and stronger attack the next time the antigen invades. This is called immunological memory. During this secondary immune response, antibodies exist in the body for a much longer
T cells are required to search out these foreigners and identify them for destruction. T cells depend upon other cells to present antigenic fragments to them, whereas B cells can search out and recognize whole antigens without much help. T cells mature in the thymus. Two major T cells result: helper T cells and cytotoxic T cells. These are distinguished based on the glycoprotein exhibited on their outer membranes. Helper T cells exhibit the CD4 glycoprotein and cytotoxic T cells exhibit CD8 glycoprotein. Not all CD4 cells are helper T cells and not all CD8 cells are cytotoxic T cells. Helper T cells release cytokines--chemical messengers that signal growth, differentiation and the action of other immune cells like macrophages. Helper T cells also help B cells to grow and develop antibodies more quickly. Cytotoxic T cells patrol the body looking for and can destroy pathogenic cells directly, including cancerous cells. Cytotoxic T cells attach to the compromised cell and then release chemical factors that either help lyse the cell or induce programmed cell death, apoptosis.
What does the Gram stain do?
The Gram stain is a differential method of staining used to assign bacteria to one of two groups (gram-positive and gram-negative) based on the properties of their cell walls
Describe what the indole test indicates
The indole test is a biochemical test performed on bacterial species to determine the ability of the organism to convert tryptophan into indole. -bacteria that contain the enzyme tryptophanase can use tryptophan as a subtrate and break it down into its metabolic products, namely, indole, pyruvic acid, and ammonia. -tryptophan is an essential amino acid that is found in nearly all proteins -looking specifically for the ability of the organism to degrade the amino acid tryptophan and produce indole
Describe the lymphatic components in organs -peyers patches monitor the bacterial load in the intestines-they capture and destroy bacteria in tremendous numbers in the intestine preventing them from penetrating the intestinal wall -spleen controls the amount of blood,the cells,and platelets in the body-blood-rich organ that filters blood. filters and cleanses blood of bacteria, viruses, and other debris. its most important function is to destroy worn-out red blood cells and return some of the breakdown products to the liver Thymus-functions at peak levels only during youth, is a lymphatic mass found low in the throat overlying the heart. produces hormones but functions in the lymphatic system by programing certain lymphocytes so they can carry out protective roles in the body Tonsils-small masses of lymphatic tissue that ring the pharynx, where they are found in the mucosa. their job is to trap and bacteria or other foreign pathogens entering the throat Bone Marrow-a key component of the lymphatic system, producing the lymphocoytes that support the body's immune system Lymphatic Vessels-pick up excess tissue fluid, called lymph and return it to the bloodstream Lymphnodes-house lym
The lymphatic system, or lymphoid system, is an organ system in vertebrates that is part of the circulatory system and the immune system. It is made up of a large network of lymphatic vessels, lymphatic or lymphoid organs, and lymphoid tissues. Returns excess interstitial fluid to Cardiovascular System Provides defense against infectious diseases and cancer Harbors leukocytes Absorbs dietary lipids from digestive tract
What types of genes (functionalities) do plasmids contain
These plasmids contain genes that code for bacteriocins, proteins that can kill other bacteria.=DNA /non-essential genes contains genes that are important to specific situational and environmental factors. Examples: Antibiotic resistance gene, & utilization of unusual substrates.
Why must a slide be air-dried and heat-fixed before staining
This distorts the cells, give improper representation of the cells shape/size Air drying reduces cell shrinkage BEFORE heat fixation
Why are controls used in the Gram stain
To ensure procedure was done correctly As a Positive Control we use a known Gram-positive bacteria. In this simulation, Staphylococcus aureus is used. If the Gram stain is performed properly, the positive control should appear purple (Gram-positive). As a Negative Control we use a known Gram-negative bacteria. In this simulation, Escherichia coli is used. If the Gram stain is performed properly, the negative control should appear pink (Gram-negative).
-ON PRACTICAL -WILL HAVE TO TELL THE PROCEDURE -RATIONALE BEHIND THE PROCEDURE -WILL BE PRESENTED WITH SCENARIOS ON WHAT COULD HAVE GONE WRONG
What can go wrong? Technique used: can result in the organisms not being able to retain the complex ---Over-heating during heat fixation ---Over-decolorizing EX-What will happen if we over decolorize-if we put the decolorizer on for a minute instead of a couple seconds =it will leach out the color on gram positive cells-so when you look at the slide under the microscope all of your cells will be pink-both the cocci and the bacillus ---Over-staining ---Too much washing between steps •Age of the culture: older organisms may lose their ability to retain the complex (cultures <24hrs) •The organism itself: some Gram Positive organisms can retain the complex better then others
selective toxicity
a chemical agent or drug can exert a toxic effect on a pathogen and leave the infected host organism unharmed -an agent is only toxic to microbial cells bc it targets a specific cell component that is only found in that specific type of microbe/and not other microbes that are in the hosts cell
What 2 genuses (types) of bacteria is the acid-fast stain used to detect?
a differential stain used to identify acid-fast bacterial organisms bacteria can be classified as either acid fast +/acid fast - genus Mycobacterium and Nocardia are acid fast +
acid fast stain
a differential stain used to identify bacteria that are not decolorized by acid-alcohol
Describe what a plasmid is
a small, circular, double stranded DNA molecule, which can replicate independently from its chromosomal DNA -genetic material thats able to self replicate -not apart of the normal DNA within the cell -mostly circular types of DNA that we see within bacteria -importantly dangerous for microbial resistance-transferred via the plasmid
a. What does the counter-stain do
a stain used to color parts of a microscopy specimen not affected by another stain especially Uses a Safranin stain it stains any colorless cells (gram negative) (PINK)
1. A student makes a smear by applying a LARGE amount of bacteria onto the slide. A simple stain is performed, but when the student looks under the microscope, individual organisms can't be distinguished. What did the student do wrong when making the smear?
did not smear it thin enough making it to thick for light to pass through and making it to hard to distinguish between what you're looking at **primary reason** she did not heat fix the slide
a. What is used for decolorization? What happens during the decolorization?
process of removing brightly colored organic impurities from the sample mixture. 95% alcohol is used for decolorizer Leaches the dye mordant complex out of the gram negative cells; decolorizing them Gram positive cells are still able to retain the dye-mordant
What color are Gram positive organisms
purple
A student mixes a known gram positive organism onto the same slide to make a smear, then performs a gram stain on the slide. When she observes it under the microscope, only gram negative organisms are seen. What did the student do wrong? (she did not forget the primary stain!)
switching the order of steps 2 and 3 She forgot to add the counterstain -she didnt add the iodine -she decolorized for too long (disrupt the bacterial cells/unable to hold the stain/wont see them anymore)
