Print This
Introduction
index |
page 1 of 40
This is the EPA
Method 1622/1623 Microscopy module on
Giardia and
Cryptosporidium. The U.S. Environmental Protection Agency (EPA) is required to evaluate the risk to public health posed by drinking water contaminants, including waterborne parasites such as
Giardia and
Cryptosporidium. Method 1622/1623 uses filtration,
immunomagnetic separation (IMS) and microscopy to detect
Giardia and
Cryptosporidium. This module was developed to aid new and experienced
analysts in the proper identification and characterization of
Giardia and
Cryptosporidium. This module also targets the characteristics of interfering organisms.
narration: Welcome to the EPA Method 1622/1623 microscopy module on Giardia and Cryptosporidium. Method 1622/1623 is the most widely used method in the U.S. for detecting these specific parasites in water. Method 1622 detects Cryptosporidium only, whereas, Method 1623 was developed to address utilities concerns regarding the presence of Giardia as well as Cryptosporidium. Both methods use microscopy as the means for identification and characterization. Therefore, the ability to properly identify Giardia and Cryptosporidium and distinguish both organisms from interfering organisms is critical to the proper implementation of Method 1622/1623.
Lesson Information
crypto0010 |
page 2 of 40
Identification and enumeration by microscopy is the determinative technique of Method 1622/1623. Narration, photographs and video are used to explain key diagnostic features of both Giardia and Cryptosporidium. After examining the positive specimens, possible variations and interfering organisms will be considered.
Knowledge reviews and summaries will help highlight key points. After two attempts of each Knowledge Review, correct answers and explanations will be given; however, the Knowledge Reviews can be answered and reviewed as many times as needed. Credit for each photograph is indicated with a letter which refers to the photographer listed on the acknowledgement page at the end of lesson.
This module is best viewed with a minimum of 800x600 screen resolution. Macromedia Flash 7 is required to view interactive graphics and video segments. Photographs or the complete module may be printed using individual print icons or through the Resources link. The narration text may be viewed by clicking on the capital T in the menu bar. Please make sure the computer audio is turned on if you wish to hear the narration.
narration: Microscopy is a critical skill for analysts performing Method 1622/1623. This module will help guide the analyst through the process of Giardia and Cryptosporidium characterization. There will be an opportunity to simulate focusing through an organism to see the series of planes visible at the microscope. Diagnostic features of positive specimens, possible variations and unknown specimens are covered. Observation of the diagnostic features depends on the cleanliness and proper alignment of the microscope, especially Köhler illumination. This module is not intended to substitute for time and experience at the microscope but to supplement it.
Lesson Objectives
crypto0020 |
page 3 of 40
After completing this module and additional practice you should be able to:
- Detect Giardia and Cryptosporidium by fluorescence (FITC-fluorescein isothiocyanate)
- Describe nucleic acid material stained by 4',6-diamidino-2-phenylindole (DAPI)
- Describe the internal morphology of targeted organisms using differential interference contrast microscopy (DIC)
- Measure Giardia cysts and Cryptosporidium oocysts
- Characterize the variations observed in Giardia and Cryptosporidium
- Identify the characteristics of interfering organisms
narration: On completion of this module and subsequent practice, participants will be able to identify and describe Giardia and Cryptosporidium as defined in Method 1622/1623. The objectives listed highlight the characteristics of Giardia and Cryptosporidium and the variations observed, contrasting these characteristics with those of interfering organisms.
Introduction to FITC, DAPI and DIC
crypto0030 |
page 4 of 40
FITC (fluorescein isothiocyanate) refers to the fluorochrome used to label the monoclonal antibodies targeted against the cell wall antigens of both Giardia and Cryptosporidium. FITC fluoresces a brilliant apple green color. The color produced by FITC is superimposed on the wall of the cyst/oocyst.
DAPI (4’,6-diamidino-2-phenylindole) is a nucleic acid stain. It fluoresces blue and can show the position and number of nuclei present in the cyst/oocyst. The absence of DAPI stained nuclei may mean nuclei are absent or nuclei are present but the stain did not come in contact with the nuclei, possibly due to stain exclusion by the cell wall.
DIC (differential interference contrast) microscopy approximates a 3-D view of the object using polarized light which allows the microscopist to view the internal morphology or structure and note any unusual characteristics.
narration: Throughout this training module, Giardia and Cryptosporidium are characterized in three ways: FITC, DAPI and DIC. These three terms refer to the stains and respective microscope settings used to view the cysts and oocysts resulting in different observable characteristics. FITC and DAPI use UV light with different filter cubes, whereas DIC uses transmitted light. To make a decision whether an object is Giardia or Cryptosporidium or an interfering organism, the microscopist uses information from all three settings.
Introduction to FITC, DAPI and DIC
crypto0040 |
page 5 of 40
The video on the right shows both Giardia cysts (single organism in the center) and Cryptosporidium oocysts (organisms below cyst) by FITC, DAPI and DIC integrated into one view. Note that the Giardia cyst exhibits distinct DAPI stained nuclei which are not observed in the Cryptosporidium oocysts. The video on the left shows the same organisms by DIC only. Use the slider or the arrows on the control bar to simulate focusing through the organisms.
swf*
swf*
narration: Use the slider or the arrows on the control bar with each video to simulate focusing through the organisms. These video sequences were produced and enhanced using special software. The video on the right shows FITC, DAPI, and DIC of both Giardia and Cryptosporidium; the video on the left shows DIC only. Review both videos as many times as necessary to note the FITC, DAPI, and DIC. Note the placement of the median bodies and nuclei in the cyst. While focusing in and out, notice how the internal features of the oocysts become apparent. Compare the placement of FITC and DAPI fluorescence to the structures observed on DIC only.
Characterization of Giardia: FITC
crypto0050 |
page 6 of 40
Accurate and precise detection is imperative for pathogen enumeration.
- Brilliant apple-green color
- Brightly highlighted rim
- Fluorescence of rim is crisp and sharp
- Fluorescence of rim is consistent width around the entire organism
- Interior fluorescence dimmer than rim
- No scattering of fluorescence when focusing
- One fluorescing rim is observed
- 5-15 µm wide, 8-18 µm long
- Correct size and shape – round to ovoid
Record observations on the Giardia examination results form.
Tricks and Tips
narration: The wall of Giardia cysts are stained with FITC labeled antibodies. A properly stained organism of the correct size and shape is the first indication that the organism may be Giardia. The fluorescence is crisp and sharp, highlighting the edge of the cyst. As the analyst focuses up and down through the organism, the fluorescence on the edge stays uniform; it does not scatter as focusing occurs. The shape may be round to ovoid depending on the orientation of the cyst. Record the shape in the appropriate column of the Giardia Examination Results Form.
Giardia Examination Results Form
crypto0050a |
page 6a of 40
Tricks and Tips
crypto0050b |
page 6b of 40
- To reduce fading of the FITC stain and preserve the filters of the microscope, always close the shutter between the mercury bulb and the filters when not viewing the cyst.
- Always use the appropriate mounting medium for the FITC stain, otherwise the fluorescence may fade more quickly.
- Use the minimum amount of non-fluorescing immersion oil necessary to resolve the fluorescence.
- To help ensure optimal contrast and resolution, clean all parts of the microscope following the procedure described in Method 1623.
Characterization of Giardia: DAPI
crypto0060 |
page 7 of 40
The use of DAPI aids in the characterization of Giardia and interfering organisms. A DAPI positive characterization assists with identification, whereas a DAPI negative characterization does not. (Roll over each bar to view the characteristics and photographs.)
swf*
Record observations on the Giardia examination results form.
Tricks and Tips
narration: A Giardia cyst contains four nuclei which can be stained with DAPI, producing distinct areas or dots of blue fluorescence. The dots of fluorescence will be distinct from the rest of the internal blue fluorescence confirming the presence and location of a nucleus. A Giardia cyst may be classified three ways based on the results of the DAPI stain. Record DAPI descriptions in the appropriate columns on the Giardia Examination Results form. Roll over each bar to view the characteristics and photographs.
Giardia Examination Results Form
crypto0060a |
page 7a of 40
Tricks and Tips
crypto0060d |
page 7d of 40
- To reduce fading of the DAPI stain and preserve the filters on the microscope, always close the shutter between the mercury bulb and the filters when not viewing the cyst.
- Occasionally, the intense fluorescent staining of DAPI may appear white.
- When viewed with DAPI filters, the FITC fluorescing rim will be nonexistent or appear light green. Other colors are atypical.
- Experienced analysts find characterization at 1000X is necessary, even though a minimum of 400X is required by Method 1623.
- No significant correlation between viability and DAPI staining has been scientifically established.
Characterization of Giardia: DIC
crypto0080 |
page 8 of 40
Differential Interference Contrast microscopy (DIC) enhances visualization of the internal morphology of Giardia cysts and helps the microscopist discern any atypical features of a fluorescing object the size and shape of Giardia. Use 1000X magnification with a nonfluorescing immersion oil for all DIC characterizations.
swf*Record observations on the
Giardia examination results form.
Tricks and Tips
narration: The internal morphology of a Giardia cyst consists of four nuclei, two axonemes and two median bodies. DIC allows the microscopist to observe any or all of these internal structures if they are present and discernable. A cyst may be categorized as empty or amorphous if the internal structures are not observed. Roll over each bar to view the characteristics and photographs.
Giardia Results - DIC
crypto0080a |
page 8a of 40
DIC observations are recorded in the 5 DIC columns on the Giardia Examination Results Form. Median bodies and axonemes are recorded as presence/absence; whereas the numbers of nuclei are recorded.
Tips and Tricks
crypto0080e |
page 8e of 40
- Make sure microscope is adjusted for Köhler illumination.
- Focus up and down through the organism a number of times while adjusting the prism to observe the internal morphology completely. The prism should be adjusted through the mid-point (or darkest) point to observe the structures with light cast from multiple directions.
- Adjusting the amount of light using the stage diaphragm and light source rheostat helps by altering the contrasting appearance of the internal features of the cell.
- Use the location of the DAPI stained nuclei to help find the corresponding nuclei on DIC.
- Sometimes the median bodies will be highlighted by DAPI. Use this to help locate the structures on DIC.
- When using DIC microscopy, the background color varies depending on the filters in place. Gray to blue is the typical color used.
- Always use a clean microscope and the minimum amount of non-fluorescing immersion oil necessary to ensure optimal contrast and resolution.
Knowledge Review: Giardia
crypto0110 |
page 9 of 40
Select all the characteristics observed in any of the 3 photos which indicate that each organism is Giardia. (The photos do not depict the same organism.).
narration: Select the characteristics from the list which indicate that the organisms are Giardia cysts. Select the “Check Answers” button when finished.
Characterization of Cryptosporidium: FITC
crypto0130 |
page 10 of 40
Accurate and precise detection is imperative for Cryptosporidium oocyst enumeration.
- Brilliant apple-green color
- Brightly highlighted rim
- Interior fluorescence dimmer than rim
- No scattering of fluorescence when focusing
- One fluorescing rim is observed
- Correct size and shape – 4-6 µm; spherical to ovoid
Record observations on the Cryptosporidium Examination Results Form.
Tricks and Tips
narration: The wall of Cryptosporidium oocysts is stained with FITC labeled antibodies. A properly stained organism which is the correct size and shape is the first indication that the organism may be Cryptosporidium. The fluorescence is crisp, highlighting the wall of the oocyst. As the analyst focuses through the organism, the fluorescence stays consistent; it shows no irregularities in intensity. Record the shape in the appropriate column of the Cryptosporidium Examination Results Form.
Cryptosporidium Examination Results Form
crypto0130a |
page 10a of 40
Tips and Tricks
crypto0130b |
page 10b of 40
- To reduce fading of the FITC stain and preserve the filters of the microscope, always close the shutter between the mercury bulb and the filters when not viewing the oocyst.
- Always use the appropriate mounting medium for the FITC stain, otherwise the fluorescence may fade more quickly.
- Use the minimum amount of non-fluorescing immersion oil necessary to resolve the fluorescence.
- To ensure optimal contrast and resolution, clean all parts of the microscope following the procedure described in Method 1623.
Characterization of Cryptosporidium: DAPI
crypto0140 |
page 11 of 40
The use of DAPI aids in the characterization of Cryptosporidium and interfering organisms. A DAPI positive characterization assists with identification, whereas a DAPI negative characterization does not.
swf*
Record observations on the Cryptosporidium examination results form.
Tricks and Tips
narration: A Cryptosporidium oocyst contains four sporozoites, each with a nucleus which may stain when exposed to DAPI. If stained, a distinct dot of fluorescence will confirm the location of a nucleus. An oocyst may be classified in three ways after DAPI staining. Record DAPI descriptions in the appropriate columns on the Cryptosporidium Examination Results form. Roll over each bar to view the characteristics and photographs.
Cryptosporidium Examination Results Form
crypto0140a |
page 11a of 40
Tips and Tricks
crypto0140d |
page 11d of 40
- To reduce fading of the DAPI stain and preserve the filters on the microscope, always close the shutter between the mercury bulb and the filters when not viewing the oocyst.
- Some analysts have noted that DAPI staining of Cryptosporidium usually is DAPI negative or DAPI positive with nuclei stained; DAPI positive with intense blue internal staining is observed infrequently.
- Occasionally, the intense fluorescent staining of DAPI may appear white.
- When viewed with DAPI filters, the FITC fluorescing rim will be nonexistent or appear light green. Other colors are atypical.
- Experienced analysts find characterization at 1000X is necessary, even though a minimum of 400X is required by Method 1623.
- No significant correlation between viability and DAPI staining has been scientifically established.
Characterization of Cryptosporidium: DIC
crypto0160 |
page 12 of 40
Differential Interference Contrast microscopy (DIC) enhances the visualization of the internal morphology of Cryptosporidium oocysts and helps the microscopist discern atypical features of an interfering organism. Use 1000X magnification with a nonfluorescing immersion oil for all DIC characterizations.
swf*Record observations on the
Cryptosporidium examination results form.
Tricks and Tips
narration: The internal morphology of a Cryptosporidium oocyst consists of four sporozoites. DIC allows the microscopist to observe these internal structures if they are present and discernable. A sporozoite will appear as a banana or crescent-shaped structure. The sporozoites are not in a specific position inside the oocysts, making them difficult to discern. An oocyst may be categorized as empty or amorphous if the sporozoites are not observed. Roll over each bar to view the characteristics and photographs.
Cryptosporidium Examination Results Form
crypto0160a |
page 12a of 40
DIC observations are recorded in the 3 DIC columns on the Cryptosporidium Examination Results Form.
- Empty and Amorphous are recorded as presence/absence
- Internal Structures are recorded as the number of sporozoites
Tips and Tricks
crypto0160d |
page 12d of 40
- Make sure microscope is adjusted for Köhler illumination.
- Be sure to focus through the oocyst a number of times while adjusting the prism to observe the internal morphology completely. The prism should be adjusted through the mid-point (or darkest) point to observe the structures with light cast from multiple directions.
- Adjusting the amount of light using the stage diaphragm and light source rheostat helps by altering the contrasting appearance of the internal features of the organism.
- Use placement of the DAPI stained nuclei to help distinguish placement of sporozoites.
- Distinguishing between a sporozoite and convolutions of the cell wall, distinguishing sporozoite placement and identifying sporozoites takes hours of practice using fresh organisms. Don't get discouraged.
- Try to consult with other experienced microscopists to verify structures observed. Comparing to photographs is also helpful.
- When using DIC microscopy, the background color varies depending on the filters in place. Gray to blue is the typical color used.
- Always use a clean microscope and the minimum amount of non-fluorescing immersion oil necessary to ensure proper contrast and resolution.
Giardia and Cryptosporidium Measurement under DIC
crypto0170 |
page 13 of 40
All size measurements must be determined using 1000x magnification under DIC and reported to the nearest 0.5 micrometers (µm).
- Superimpose the ocular micrometer over the (oo)cyst.
- Place the left edge of the widest point of the (oo)cyst on a line of the micrometer.
- Count the number of units to the right edge of the
(oo)cyst. - Convert the number of units counted to µm using the microscope conversion factor previously determined (see section 10.3.5 of Method 1622/1623: Calibration of an ocular micrometer). In these examples, one unit on the ocular micrometer is 1µm.
- Repeat for the longest point of the (oo)cyst.
Select the link to view sample examination results forms.
narration: Giardia cysts are round to ovoid objects 5-15 µm wide and 8-18 µm long. Cryptosporidium oocysts are ovoid or spherical objects 4-6 µm in diameter. Perform measurements of the (oo)cyst while the microscope is adjusted for DIC illumination. If debris obscures visualization of the (oo)cyst, perform measurements with the microscope adjusted for FITC.
Giardia and Cryptosporidium Examination Results Forms
crypto0170a |
page 13a of 40
Knowledge Review: Cryptosporidium
crypto0190 |
page 14 of 40
Match all observable characteristics with each photograph (each characteristic may be used once, more than once, or not at all). The photos do not depict the same organism. No commas or spaces are necessary between the numbers if more than one characteristic applies to a photo.
- One sporozoite
- Correct shape
- Amorphous internal structure
- DAPI negative
- Nuclei DAPI stained
- Brilliant apple-green fluorescence
- Three sporozoites
narration: Three Cryptosporidium oocysts are shown in the photographs. Match the characteristics from the list with each photo. Select the “Check Answers” button when finished.
Review of FITC, DAPI and DIC
crypto0210 |
page 15 of 40
The video on the right, produced with special imaging equipment and software, shows a group of Cryptosporidium oocysts by FITC, DAPI and DIC integrated into one view. The video on the left shows the oocysts by DIC only. The DAPI and DIC are not as distinct as analysts would like to see due to the nature of the organisms, staining, and the software at the time the video was captured. Use the slider or arrows below the video to focus through the organisms as many times as you wish.
swf*
swf*
narration: Using enhancement by special software, the video on the right shows FITC, DAPI and DIC of a group of Cryptosporidium oocysts integrated into one view. DIC only is shown in the video on the left. Review both videos as many times as necessary to note the FITC, DAPI and DIC. Be sure to note the placement of the FITC stain superimposed on the wall shown on DIC and DAPI highlighting the nuclei in one end of a sporozoite. Note also the sporozoite placement in the oocysts.
Variation of Giardia: FITC
crypto0220 |
page 16 of 40
- Fluorescing lines inside the wall – wrinkles
- Fluorescing lines inside the wall – edge of cytoplasm fluoresces
- Fluorescing wall is curved – cyst folded or bent
narration: Now that you have seen how Giardia should fluoresce with FITC, it’s time to look at the possible variations. The fluorescence can appear different between cysts within a sample and between samples. Cysts vary in condition which will influence the fluorescent pattern. Additional fluorescing lines may be observed when the cyst is wrinkled or folded. Some cysts will exhibit fluorescence at the edge of the cytoplasm. Spend the time to become familiar with the Giardia on the positive control slide, the On-going Precision and Recovery (OPR) slide and in waters you regularly analyze; these subtle variations will become apparent.
Variation of Giardia:DAPI
crypto0230 |
page 17 of 40
- Variation in intensity and color of stained nuclei
– white to blue - Variation in size and shape of stained nuclei
– round to oval - Fluorescence of cyst wall
– color
*Reminder: Depending on the filters used and the microscope configuration, the FITC fluorescing rim will be nonexistent or appear light green. Other colors are atypical. Review "Tricks and Tips" on Screen 7.
narration: Like FITC, DAPI may exhibit different characteristics between cysts in a sample and between samples. Moreover, the concentration of DAPI may be adjusted during sample processing by Method 1623 in order to accommodate for fading or diffusion of the DAPI stain. Note the variation in DAPI stain in these photographs. Many observations of the DAPI intensity and wall characteristics of cysts on the positive control and OPR slides are necessary to learn all the possible variations.
Variation of Giardia:DIC
crypto0240 |
page 18 of 40
- Wrinkles or folds
- Cytoplasm – granular
- Cytoplasm – separated from wall
- Wall condition – may appear fragile
Roll over each variation to view the corresponding FITC.
swf*
narration: The morphology of a Giardia cyst varies depending on it's condition. The cell wall may fold, wrinkle or appear fragile as the cyst degrades. Similarly, the nuclei, axonemes and median bodies may also degrade and disappear. The cytoplasm may appear granular or brownish or may pull away from the cell wall forming an empty space. Look at lots of live Giardia, which have been exposed to a variety of conditions, to see the range of variation in the internal structures and the walls. Don’t always look for the “perfect” specimens – examining the empty and amorphous cysts helps reveal the range of variation possible.
Knowledge Review: Giardia
crypto0250 |
page 19 of 40
Mark the characteristic(s) which is(are) depicted in the DIC and/or DAPI photographs of the same organism:
narration: Select the characteristics which are observed in both photographs of Giardia; then select the "Check Answers" button.
Variation of Cryptosporidium:FITC
crypto0260 |
page 20 of 40
- Wrinkled – fluorescing lines inside wall
- Ruptured – “PacMan” variety, may be partially
excysted
- Folded – punched in ball, open clam look
narration: The variations in the appearance of the cell wall of Cryptosporidium with FITC usually relate to whether or not the oocyst is intact or the osmotic pressure of the solution in which the oocysts were suspended. The oocysts may appear wrinkled or if the sporozoites have excysted, the oocyst will appear popped open exhibiting a “PacMan” look. Oocysts will also fold – forming various shapes such as an opened clam or a punched in ball.
Variation of Cryptosporidium:DAPI
crypto0270 |
page 21 of 40
- Variation in intensity and color of stained nuclei
– white to blue - Variation in size and shape of stained nuclei
– round to oval - Fluorescence of cyst wall
– color
*Reminder: Depending on the filters used and the microscope configuration, the FITC fluorescing rim will be nonexistent or appear light green. Other colors are atypical. Review "Tricks and Tips" on Screen 11.
narration: Most analysts note that DAPI staining for Cryptosporidium is quite consistent with regard to size and shape of nuclei stained. However, the intensity of the fluorescence, either nuclei stained or overall internal staining, may vary between oocysts. Many times it is difficult to see all four nuclei stained by DAPI because they are located in different planes. Careful focusing through the oocysts to view the multiple planes is imperative. To learn all the possible variations in the DAPI intensity and wall characteristics of oocysts, continually review the positive control and OPR slides. Remember that the concentration of DAPI may be adjusted in Method 1622/1623 (Section 14.6) if fading or diffusion of the DAPI stain is noted.
Variation of Cryptosporidium:DIC
crypto0280 |
page 22 of 40
- Wrinkled
- Ruptured – “PacMan” variety, may be partially excysted
- Folded – punched in ball, open clam look
- Sporozoite placement
Roll over each variation to view corresponding FITC.
Select the link to view a video sample of multiple oocysts.
narration: The variations observed in Cryptosporidium on DIC parallel the variations noted with FITC. Folded or wrinkled oocysts may appear to have additional internal structures; don't mistake a fold or wrinkle for a sporozoite. Folds and wrinkles usually fluoresce on FITC but sporozoites do not fluoresce. Remember that the sporozoites are positioned inside a sphere, so you may only be able to see the ends of the sporozoites. Use the position of the DAPI stained nuclei to narrow-down the location of the sporozoites. Again, spend time observing many Cryptosporidium oocysts on the positive control, OPR slide and field samples to learn the possible variations.
Variations of Cryptosporidium Sporozoites
crypto0280v |
page 22v of 40
View the video sample for sporozoites in the oocysts. Sporozoites are the most distinct in the three oocysts marked by arrows.
Knowledge Review: Cryptosporidium
crypto0290 |
page 23 of 40
Select all diagnostic features which indicate the organisms in the photographs are possible Cryptosporidium.
(The photographs do not depict the same organism. Assume the microscope is adjusted for maximum contrast and resolution.)
narration: The photographs depict three possible Cryptosporidium oocysts. Choose the diagnostic features observed that indicate the organisms are oocysts.
Characterization of Interfering Organisms
crypto0300 |
page 24 of 40
It is essential that analysts learn to recognize cross-reacting organisms as well as the range of variation of Giardia cysts and Cryptosporidium oocysts to accurately and consistently identify these pathogens. Below are general features which may indicate that the observed organism is not Giardia or Cryptosporidium, but a cross-reacting or auto-fluorescing organism:
- Irregular fluorescence
- Different size and/or shape
- Irregular DAPI staining
- Atypical structures on DIC
narration: The first section of this module covered how Giardia or Cryptosporidium should look with FITC, DAPI and DIC. This section will highlight features which indicate organisms are not Giardia or Cryptosporidium. Some features such as visible spines on DIC are quite obvious; other features are quite subtle to detect. An analyst needs to use information gathered from sample processing and examination to make the most informed decision. Determining cross-reacting organisms is a time consuming proposition but very necessary for proper enumeration of cysts and oocysts in an environmental sample.
Interfering Organisms: Giardia-like FITC
crypto0310 |
page 25 of 40
The following are features of organisms observed with FITC fluorescence which help the analyst to differentiate the organism from Giardia:
- Variation in intensity and color of fluorescence
- Even fluorescence over entire Giardia-like organism
- Atypical width of fluorescing rim
- Breaks in fluorescence
- More than one fluorescing wall
- Incorrect shape and size
narration: Giardia FITC fluorescence varies between cysts. Additional fluorescing lines may be observed when the cyst is wrinkled or folded. Even though there is variation in fluorescence of cysts, certain features indicate interfering organisms. These features include fluorescence that is not sharp, not superimposed on the wall, and inconsistent or irregular. When scanning environmental samples, examine all possible fluorescing objects to learn how interfering organisms fluoresce.
Interfering Organisms: Giardia-like DAPI
crypto0320 |
page 26 of 40
The following are features of organisms observed with DAPI fluorescence that indicate the organism is not Giardia:
- More than four DAPI stained nuclei
- One large DAPI stained nuclei
- Atypical color of DAPI
- Atypical placement of DAPI stained nuclei
- Other colors inside the cyst
- Atypical wall color
swf*
narration: Ideally, Giardia DAPI would show four intense blue dots. When the DAPI does not show four nuclei, it may be due to the wall impermeability or the absence of nuclei. However, this does not mean that the organism is not Giardia. It is critical to focus through the organism to distinguish the nuclei stained from the background DAPI fluorescence and separate any distinct nuclei. Cyst-like objects with one large DAPI dot, more than four DAPI dots or other colors on DAPI are indications the object is not Giardia. Roll over the last four photos to view the corresponding FITC photos.
Interfering Organisms: Giardia-like DIC
crypto0330 |
page 27 of 40
Observations on DIC that help distinguish an interfering organism from Giardia:
- The wall observed on DIC is not superimposed by the FITC fluorescing wall
- More than one wall
- Variations in wall width
- Openings or breaks in wall
- Internals composed of small spheres
| - Internals composed of more than one large distinct mass
- Organism composed of multiple cells
- Presence of
mucilage around the organism
- Atypical shape and size
- Presence of colored internal structures
|
swf*
narration: Ideally with DIC, the analyst observes a cell wall, nuclei, median bodies and axonemes for each cyst. Cross-reacting organisms will often exhibit irregular, inconsistent, glassy or heavy walls. Atypical internal morphology may consist of small distinct spheres or other structures not consistent with Giardia. Carefully examine the placement of the FITC fluorescence to see if it corresponds to the wall observed on DIC. Also the placement of the DAPI dots, if present, helps in locating the corresponding nuclei. Roll over each photo to view corresponding DAPI and FITC photos.
Knowledge Review: Giardia
crypto0335 |
page 28 of 40
narration: Is the organism shown in the three photographs Giardia? Select all the characteristics observed that support a yes or no answer, then select the “Check Answers” button.
Interfering Organisms: Cryptosporidium-like FITC
crypto0340 |
page 29 of 40
- Variation in intensity and color of fluorescence
- Even fluorescence over entire Cryptosporidium-like organism
- Uncharacteristically narrow or wide fluorescence
- Inconsistent intensity of fluorescence
- Breaks in fluorescence
- Incorrect shape and size
Select the link to view more comparisons.
narration: The fluorescence of Cryptosporidium oocysts varies in intensity but is always crisp and sharp. The presence of a ruptured cyst (“PacMan” look) is also diagnostic of Cryptosporidium oocysts. Cross-reacting organisms often exhibit fluorescence that scatters or does not remain sharp even if it is brilliant apple-green. Some common cross-reacting organisms will exhibit round openings in the fluorescence different from the typical ruptured opening. Select the link to view additional photographs of interfering organisms.
Cryptosporidium-like FITC
crypto0340a |
page 29a of 40
Compare the typical FITC fluorescence pattern to the FITC fluorescence of interfering organisms.
Interfering Organisms: Cryptosporidium-like DAPI
crypto0350 |
page 30 of 40
- More than four DAPI stained nuclei
- DAPI stained nuclei are too large
- Atypical color of DAPI
- Other colors inside the cyst
- Atypical wall color
Roll over each photo to view corresponding FITC.
Select the link to view more comparisons.
narration: Cryptosporidium oocysts may exhibit four small but distinct DAPI stained nuclei. Cross-reacting organisms often contain more than four DAPI dots or one DAPI dot which fills the entire oocyst-like organism. In cross-reacting organisms, the stained nuclei may not be well-differentiated but more diffuse. Interfering organisms may also exhibit other colored organelles inside the organism. Select the link to view positive control Cryptosporidium oocysts and additional cross-reacting organisms.
Cryptosporidium-like DAPI
crypto0350a |
page 30a of 40
Compare the typical DAPI positive pattern to the DAPI pattern of interfering organisms.
Interfering Organisms: Cryptosporidium-like DIC
crypto0360 |
page 31 of 40
- FITC fluorescing wall not superimposed on the wall observed on DIC
- Absence of wall or more than one wall
- Uncharacteristically narrow or wide wall
- Openings or breaks in wall, unlike "Pac Man" structure
| - Oocyst-like organism subdivided into multiple sections or cells
- Presence of mucilage
- Atypical shape and size
- Presence of colored internals
|
|
Roll over each photo to view corresponding DAPI and FITC. Select the link to view more
comparisons.
narration: The sporozoites in Cryptosporidium oocysts are difficult to discern. Therefore, recognition of atypical internal morphology, not the absence of sporozoites, is important for the analyst. Cytoplasm which does not separate from the oocyst wall as well as the presence of one large nucleus is indicative of a cross-reacting organism. Interfering organisms often display inconsistent walls. Select the link to view other comparisons. Examining a variety of organisms in environmental samples will improve an analyst's ability to discern atypical characteristics.
Cryptosporidium-like DIC
crypto0360a |
page 31a of 40
Compare the ideal pattern of the wall and sporozoites with the patterns observed in interfering organisms.
Knowledge Review: Cryptosporidium
crypto0370 |
page 32 of 40
Is it
Cryptosporidium? If all three photos represented the same organism, support why you think the organism is or is not
Cryptosporidium by checking the characteristics observed in the photos. Once you have decided, check the Yes or No box below. (Assume the microscope is adjusted for maximum contrast and resolution.)
narration: Assume these three photographs show the same possible Cryptosporidium oocyst. Decide whether it is an oocyst or not by selecting the observable characteristics and choosing “Yes” or “No”.
Knowledge Review
crypto0380 |
page 33 of 40