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6.2: Procedure - Biology

6.2: Procedure - Biology


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Your instructor will show you an example of a simple dichotomous key using geometric shapes (see below). There are many ways to make a useful key, so your key may not resemble those made by other students.

Note

Although additional staining can sometimes be used as part of a dichotomous key, you will be only using the metabolic tests for this exercise.

Once you have completed your dichotomous key, review the gram reaction and morphologies of your two unknowns. Based on these results, decide which media should be used to identify your unknowns, and write them down in the space provided on the back of your dichotomous key. When you are finished, turn it in to your instructor. You can do this during the lab period (please make sure it is legible), or you can take it home to work on it, provided you return it to your instructor at least 3 days before your next scheduled lab. Your instructor will check it to make sure it is correct and that you have identified the correct media to use to identify your unknowns. Your key will be graded and returned to you by the next lab period when you will inoculate your chosen media with your unknowns. Points will be deducted for late dichotomous keys.


6.2: Procedure - Biology

In both meiosis and mitosis, the original parent cell is a diploid cell. Remember, this means the cell has two copies of each chromosome. Meiosis and mitosis are both nuclear divisions that result in new daughter cells. However, the two processes have significant differences. Fill out the following chart comparing the two forms of nuclear division.

Mitosis
(begins with a single cell)
Meiosis
(begins with a single cell)
# chromosomes in parent cells
# DNA replications
# nuclear divisions
# daughter cells produced
purpose

Think about It

Does mitosis and/or meiosis happen in prokaryotes? Do mitosis and/or meiosis happen in eukaryotes? Explain.


Explain Activity 6.2 NCERT class 10 science Life processes

Activity 6.2 asks us to test for starch in a plant with iodine, and compare it with the plant kept with potassium hydroxide.

Observation:
  1. Leaf of plant kept without potassium hydroxide turn blue with iodine solution.
  2. The plant with potassium hydroxide does not turn blue.
Explanation:

Potassium hydroxide is a highly reactive compound. It reacts with the carbon dioxide of the bell and forms its carbonate.

As a result, no carbon dioxide is available for the plant. In the absence of Carbon dioxide plant is unable to do photosynthesize and form starch. So, iodine does not turn the leaf blue.

The plant kept in bell without Potassium hydroxide can do photosynthesis and form starch. So, iodine solution here turns the leaf blue.

Inference/conclusion:

This experiment shows that carbon dioxide is necessary for photosynthesis and starch formation.


Procedure for Identification of Bacterial Culture

The following points highlight the four main procedure for identification of bacterial culture. The procedure are: 1. Examination of Solid Media 2. Examination of Liquid Medium 3. Primary Diagnosis 4. Final Identification/Confirmation of Bacteria.

Bacterial Culture: Procedure # 1. Examination of Solid Media:

The medium and colonial appearance of bacterial growth is to be identified. It is useful for provisional identification of bacteria. Study a Gram’s-stained smear of bacteria and observe staining reaction and microscopic morphology of bacteria.

Usually nutrient agar, blood agar or MacConkey’s agar.

2. Colony morphology (Fig. 6.2):

Pinpoint (streptococcus), pinhead (staph.), large (Klebsiella), moderately large (E. coli), Small (Shigella) etc.

Dome (Staph), flat (vibrio), low convex (Salmonella, Shigella), droughtsman (S. pneumoniae), convex (Klebsiella, E.coli) irregular (Bacillus) etc.

Rough (Some Neisseria, Bacillus), matt (E. coli), ridged (Pseudomonas) smooth (Salmonella) etc.

Entire (Salmonella), irregu­lar (Pseudomonas), crenated (Bacillus), spre­ading (Proteus) etc.

Opaque (Staph), transparent (Vibrio), semi-translucent (Salmonella).

Golden yellow (Staph aureus), bluish- green (Pseudomonas), brick-red (Serratia marcescens).

vii. Pigment restricted to colony — Staph, Micrococcus, Serratia.

viii. Pigment diffused in medium — Pseudomonas.

ix. Haemolysis — Beta haemolysis (S. pyogenes), alfa haemolysis (S. pneumoniae).

x. Haemodigestion — Proteus, Pseudomonas etc.

xi. Consistency — Mucoid (Klebsiella), butyrous (Staph), friable (some Neisseriae), firm (Bacillus), Sticky (V. parahaemolyticus).

3. Gram stained smear examination:

(i) Staining reaction:

(ii) Microscopic morphology (Fig. 6.1) — Note the following:

a. Shape— rod (E. coli etc.), spherical or cocci (Staph etc.), coccobacilli (Brucella etc.), comma (Vibrio).

b. Length/Size— long (E. coli), short (Klebsiella etc.), large (Micrococcus), small (Streptococcus) etc.

c. Width — stout (E. coli, Bacillus), slender (Salmonella) etc.

(iii) Unstained space for spore (Bacillus, Clostri­dium).

(iv) Ends — rounded (E. coli), square cut (bacillus), tapered (diphtheroid).

(v) Sides — parallel, un-parallel and irregular.

(vi) Inclusion granules.

Bacterial Culture: Procedure # 2. Examination of Liquid Medium:

i. Identify the liquid medium.

ii. Note the bacterial growth pattern.

iii. Examine the motility and morphology (if possible) in hanging drop preparation.

1. Identification of liquid culture medium:

i. Peptone water:

No tinge of colour. Most of the non-fastidious bacteria are grown and supplied in it.

ii. Glucose in nutrient broth:

All broths show straw/yellowish-blue. Usually growth of Streptococcus is supplied in this medium.

2. Bacterial growth pattern (Fig. 6.3):

i. Uniform turbidity: Example – most of the common gram negative rods.

ii. Uniform turbidity with surface pellicle formation: Examples — Strict aerobes like Vibrio, Pseudo­monas and Bacillus concentrate near the surface.

iii. Deposit at bottom: Heavy bacteria form deposit leaving slight haze above. Example — Strepto­coccus.

3. Hanging drop preparation:

i. True/active motility or non-propagative brownian movement.

ii. If motile, characteristic of motility (e.g., darting and fish in stream by Vibrio cholerae, tumbling motility by Y. enterocolitica etc.)

iii. Morphology — rods are identifiable but cocci in cluster, discrete or in short chain are difficult to visualize (Fig. 6.4).

Bacterial Culture: Procedure # 3. Primary Diagnosis:

Colony character, medium on which growth appe­ared, pattern of growth in liquid medium, gram stained morphology and motility study generate clues for primary diagnosis and help to formulate scheme for confirmatory or final diagnosis.

Bacterial Culture: Procedure # 4. Final Identification/Confirmation of Bacteria:

Sugar fermentation tests, utilisation pattern of amino acids, tests for some enzymes and metabolic pattern, ability to grow on synthetic medium, H2S production, indole production, MR-VP test, urease produ­ction, P.P.A deaminase test etc.

2. Slide agglutination test using high titre anti- sera.

3. Toxigenicity test or animal pathogenicity test.

4. Molecular method (hybridisation with DNA probe, detection of signature sequence in DNA and in 16S rRNA).


Conclusion

In our quest to create self-regulatory systems for recombinant protein production, we used an integrated synthetic biology approach to construct a synthetic circuit that limits recombinant protein production through stress-induced feedback. We validated the functionality of different variants of the synthetic circuit in their capacity to limit stressful protein production, and to increase the total soluble fraction. Since the protein yield was significantly lowered in the process, further investigation on promoter and repressor engineering for avoiding such loss would be welcomed. In addition, since different proteins lead to different levels of stress within the host cells [9], it would be interesting to test this approach with other recombinant protein species. Furthermore different inducer concentrations can be used to tune transcription rate and product formation, although there is evidence that inducer concentration does not necessarily influence the formation of active soluble protein [28].

The computational model that we constructed provided valid predictions on the system dynamics, and was useful as a first order guiding tool for our experimental design. An extended phenomenological description and inclusion of a larger set of measured experimental parameters would allow an increased predictive accuracy of the model, and it may help to test or generate alternative hypotheses regarding the the dynamics of inclusion body formation and their degradation rates. This study provides an example of how integration of computational, engineering and experimental methods, together with the synthetic biology concepts of parts standardization, can be applied to address biotechnological challenges from a new perspective. Altogether, this and similar future studies can be applied to guide the construction of robust auto-regulatory protein production systems.


Conclusions

The present study demonstrates that a combination of mRNA tagging and microarray analysis is an effective strategy for identifying genes expressed in subsets of neurons. Systematic reporter expression analyses following this approach will facilitate the accumulation of information regarding gene expression patterns. In particular, profiling of the gene expression patterns of subsets of neurons, in combination with analyses of neural functions, might provide insights into understanding the distinct roles of cells within the neural network.


Models for Assessing Anti-Angiogenesis Agents: Appraisal of Current Techniques

Subcutaneous and Orthotopic Xenografts

For studies of tumor biology and relevant angiogenesis, it is attractive to study orthotopic tumor implants (xenografts) in which the local microenvironment resembles that in human cancers [3] . Subcutaneous tumor implants are less desirable from the standpoint of modeling the condition in patients, but these are a technically convenient option for the studying of dose escalation of single drugs and for comparing effects of multiple agents. The blood vessel supply about tumor xenografts is generous. This permits histologic and biochemical analysis. In addition, a simplified index of vascularity, for example, tumor hemoglobin content, is straightforward in subcutaneous xenografts, and the technical expertise needed for many types of orthotopic insertion is avoided. Two weeks is sufficient for the establishment of most xenografts, after which drug or factor testing is initiated and continues for several weeks. Subcutaneous xenograft volumes may be estimated daily by calipers, and subsets of animals can be sacrificed periodically during treatment in order to obtain histology and biochemical information that is needed. The few weeks required for each drug or factor study yields substantial amounts of information and is consistent with needs to secure data about multiple drugs, drug dosages, or combinations of drugs ( Fig. 2.3 ). From the standpoints of angiogenesis and tumor cell biology, the subcutaneous xenograft model is a useful screening option in studies of angiogenesis.

Tumor measurements length, width, and height are feasible when papable tumors are present and this usually occurs within 7 days after subcutaneous tumor cell inoculation. However, tumor size is a function of cancer cell type. Quantitation of angiogenesis in xenografts is feasible with CD31 immunostaining, with other endothelial cell markers or by spectrophotometric estimation of hemoglobin content using Drabkin’s reagent.

Orthotopic human tumor implants in the nude mouse closely mimic the local microenvironmental conditions of the primary clinical tumor. Tumor behaviors and the activity of the associated vasculature reflect the interaction of local host factors with the intrinsic qualities of the cancer cells [19] . Interstitial pressure generation in orthotopic tumors is likely to be quite different from that achieved with subcutaneous implantation. Increased interstitial pressure of course restricts access of systemically administered drugs, including angiogenesis-relevant agents, to tumor cells. In addition, certain qualities of blood vessels are distinctive at the orthotopic site. Among such qualities are microvessel density, blood vessel permeability, and blood vessel cell gene expression [20] . Transcriptional differences among implantation sites, depending on the tumor cell type, have involved tumor genes for FGF2 (bFGF), for VEGF, and VEGF receptors and, importantly, for genes involved in the development of multidrug resistance and in supporting inflammation [3] .

The nude mouse is small and this enables serial nonradioisotopic fluorescence IVIS imaging (in vivo imaging system) of orthotopic cancer implants ( Fig. 2.4 [21] ), yielding information about tumor volumes that is more accurate than noninvasive volume estimates of subcutaneous xenografts. IVIS scans also distinguish viable cells from dead cells in tumor mass and may be used to measure other qualities of the cells in the scanned xenograft.

Figure 2.4 . Representative IVIS images taken 1 week after orthotopic implantation of prostate cancer cells (PC3-Luc) in male nude mice.

Loss of cell viability is tracked by bioluminescent color change [red (actively dividing cells) to blue].

(With permission of Springer: Mousa SA, Davis PJ. Angiogenesis Modulations in Health and Disease, Chapter 1 Angiogenesis Assays: An Appraisal of Current Techniques, 2013, p. 1–12).


What Is a Follicle-Stimulating Hormone Test?

If you have trouble getting pregnant or have problems with your ovaries or testicles, your doctor may want you to have a follicle-stimulating hormone (FSH) test.

FSH is a hormone made by your pituitary gland, a grape-sized organ that sits at the base of your brain. It helps women maintain healthy eggs and get pregnant. The hormone keeps men&rsquos sperm healthy.

Your doctor may order an FSH test to learn if your body is making too much or too little of the hormone. This can help the doctor see whether your fertility problem is due to an issue with your pituitary gland, your ovaries, or your testicles.

Here is information you can use to better understand what an FSH test is, why it&rsquos important, and what you can expect.

Who Gets an FSH Test?

If you&rsquore a woman, you might get an FSH test if you:

If you&rsquore a man, you might get an FSH test if:

  • Your partner can&rsquot get pregnant
  • You have a low sperm count, low muscle mass, or low desire for sex

Children might get an FSH test if puberty starts too late or too early.

Before the Test

For women, the result of your FSH test will vary based on where you are in your menstrual cycle. Because of that, your doctor will ask about your period and suggest that you take the FSH test at a certain time of the month.

Tell your doctor if you&rsquore pregnant since that also will change your test outcome.

Your doctor will want to know all the drugs you currently take. Some prescribed medicines, such as birth control pills, can skew your results.

How the Test Is Done

A health professional will draw blood from a vein in your arm and send it to a lab. In some cases, you might provide a urine (pee) sample instead. Because FSH levels go up and down so much, you may need to collect all of your pee over the course of 24 hours.

The Food and Drug Administration (FDA) has approved an at-home FSH urine test for women. But you still need to follow up with your doctor afterward for complete results. The test may signal that you&rsquore in or close to menopause (the end of your periods). But the at-home test can&rsquot give you information about your fertility.

Does the FSH Test Pose Any Health Risks?

The FSH blood test is safe. But like any time you have blood work done, you may:

  • Feel faint or lightheaded
  • Have a bruise, lump, or soreness where the needle went into your skin

Normal Results for Women and Men

An FSH test doesn&rsquot come out &ldquopositive&rdquo or &ldquonegative.&rdquo Instead, it reveals the amount of FSH you have, measured in international units per millimeter (IU/mL).

For women, results vary based on where you are in your monthly cycle.

  • Before release of an egg: 1.4 to 9.9 IU/mL
  • At egg release: 6.2 to 17.2 IU/mL
  • After egg release: 1.1 to 9.2 IU/mL
  • After menopause: 19 to 100 IU/mL

For men, normal results can range from 1.4 to 15.5 IU/mL

Many things like your age, gender, and health history can affect your FSH test result. Because of that, you&rsquoll need to rely on your doctor to explain what your number means. You may need more tests to check other hormones that are important for fertility. Together, these test results can give you and your doctor a clearer picture of what&rsquos going on with your health.


5.2 Standard Operating Procedures

Each PI must have written Standard Operating Procedures (SOPs) for high-risk materials and research protocols conducted in their laboratory.

Laboratory-specific SOPs are valuable research tools that supplement the Lab-Specific Hygiene Plan. The process of writing SOPs requires an individual to think through all steps of a procedure, perform a risk assessment assessing each step, and implement any relevant safety controls before beginning work. The SOP provides a written means to inform and advise researchers about hazards in their workplace, allows for standardization of materials and methods, and improves the quality of the research.

SOPs should include exposure controls and safety precautions that address both routine and accidental chemical, physical or biological hazards associated with the procedure. Hazard class SOP templates for developing new SOPs are available below.

Like the Chemical Hygience Plan (CHP), lab SOPs must be accessible to all researchers. Keeping hard copies in the lab or having them on a computer in the laboratory fulfills the accessibility requirement. SOPs developed through UHS will be posted periodically on the UHS website .

SOPs should be reviewed every three years for accuracy. If a lab procedure changes, a new risk assessment must be performed and the SOP must be modified.


Biological Substances

"An infectious substance which is transported in a form that, when exposure to it occurs, is capable of causing permanent disability, life-threatening or fatal disease in otherwise healthy humans or animals. Indicative examples of substances that meet these criteria are given in Table 3.6.D." (Dangerous Goods Regulation, 3.6.2.2.2.1)

What are the requirements when shipping Infectious Substances, Category A with UPS?

UPS will accept, Category A Infectious Substances assigned to UN2814 or UN2900, provided the following conditions are met before offering this commodity:

  • A signed agreement expressly permitting the shipment of Infectious Substance, Category A is required.
  • Service only available within the U.S. and Puerto Rico.
  • Service is not available for "Select Agents or Toxins," as regulated by the Centers for Disease Control and Prevention under 42 CFR Part 73, or the U.S. Department of Agriculture, under 9 CFR Part 21.
  • For enhanced visibility, UPS Proactive Response is required.
  • Shipments must be prepared in accordance with the IATA Dangerous Goods Regulations.
  • UPS Next Day Air ® or Next Day Air ® Early A.M. ® service must be used for Infectious Substance, Category A. No other service levels, including UPS Next Day Air Saver ® , are acceptable for this commodity.

What are the regulatory requirements that must be followed when offering Infectious Substances, Category A?

Refer to the Dangerous Goods Regulations, Packing Instruction 620 for more information regarding marking, labeling, and packing requirements.

Infectious Substances, Category B:

What is an Infectious Substance, Category B (also known as Biological Substance, Category B)?

"An infectious substance which does not meet the criteria for inclusion in Category A. Infectious substances in Category B must be assigned to UN 3373." (Dangerous Goods Regulations, 3.6.2.2.2.2)

Note: The proper shipping name of UN 3373 is Biological substance Category B.

If shipping under the 49CFR, refer to 49 USC § 173.134 for additional information.

What are the requirements when shipping Infectious Substances, Category B with UPS?

UPS will accept, Biological Substances, Category B assigned to UN3373, provided the following conditions are met before offering this commodity:

  • Verify that the destination is serviceable by using the Approved Country List, Click here to access the Approved Country List .
  • For transportation outside of the United States, an International Special Commodities (ISC) Contract is required. Contact your UPS sales representation for more information.

What are the regulatory requirements that must be followed when offering Infectious Substances, Category B?

The IATA regulations require that shipments containing Biological Substances, Category B are triple packaged according to Packing Instruction 650. Refer to the Dangerous Goods Regulations, Packing Instruction 650 for more information regarding marking, labeling, and packing requirements.

If this commodity is being offered under the 49CFR refer to 49 USC § 173.199 for more detail on marking, labeling, and packaging.

Exempt Human Specimen or Exempt Animal Specimen:

What is an Exempt Human Specimen or Exempt Animal Specimen?

"Patient specimens for which there is minimal likelihood that pathogens are present." (Dangerous Goods Regulations, 3.6.2.2.3.8)

Refer to 49 USC § 173.134 for additional information regarding domestic shipping.

What are the requirements when shipping Exempt Human Specimen or Exempt Animal Specimen with UPS?

UPS will accept, Exempt Human Specimen or Exempt Animal Specimen, provided the following conditions are met before offering this commodity:

  • Verify that the destination is serviceable by using the Approved Country List, Click here to access the Approved Country List .
  • For transportation outside of the United States, an International Special Commodities (ISC) Contract is required. Contact your UPS sales representation for more information.

What are the regulatory requirements that must be followed when offering Exempt Human Specimen or Exempt Animal Specimen?

Patient specimens for which there is minimal likelihood that pathogens are present are not subject to other provisions of the Regulations provided they are marked with the words "Exempt human specimen" or "Exempt animal specimen&rdquo and packaged according to the IATA regulations. (Dangerous Goods Regulations, 3.6.2.2.3.8)

If this commodity is being offered under the 49CFR refer to 49 USC § 173.134 for more detail on marking, labeling, and packaging.

Medical, Clinical, or Regulated Waste:

What is Medical, Clinical, or Regulated Waste?

"Medical or clinical wastes are wastes derived from the medical treatment of animals or humans or from bio-research." (Dangerous Goods Regulations, 3.6.2.1.5)

Refer to 173.134 for additional information regarding domestic shipping.

What are the requirements when shipping Medical, Clinical, or Regulated Waste with UPS?

UPS does not accept Medical, Clinical or Regulated Waste for transportation. This includes all forms of waste that might be reclassified under a different proper shipping name.

If you have any questions about shipping Infectious Substances with UPS, contact your UPS Salesperson or call the Hazardous Materials Support Center at 1-800-554-9964.


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