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Method 1622/1623 Microscopy
Giardia & Cryptosporidium


Introduction

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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. Animation



Lesson Information

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3 pictures: microscopist, Giardia and Cryptosporidium

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

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After completing this module and additional practice you should be able to:


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

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FITC, DAPI, and DIC images

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

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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.

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Characterization of Giardia: FITC

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Giardia

Accurate and precise detection is imperative for pathogen enumeration.

Record observations on the Giardia examination results form.

Note Icon 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

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Giardia Benchsheet 2 pictures, one of oval shaped Giardia and second of round shaped Giardia



Tricks and Tips

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Characterization of Giardia: DAPI

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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.

Note IconTricks 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.

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Giardia Examination Results Form

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Giardia Results Examination Form highlighting DAPI columns 2 pictures, one of oval shaped Giardia and second of round shaped Giardia



Tricks and Tips

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Characterization of Giardia: DIC

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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.

Note IconTricks 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.

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Giardia Results - DIC

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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.


Giardia Results Examination Form with empty, amorphous, median bodies and axonemes shown recorded as presence absence and number of nuclei observed as a number 2 pictures, one of oval shaped Giardia and second of round shaped Giardia



Tips and Tricks

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Knowledge Review: Giardia

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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.).


1.  Brightly highlighted rim
2.  Brilliant apple-green fluorescence
3.  Proper shape
4.  Intensity of fluorescence is similar between the rim and the interior
5.  Intense blue internal DAPI staining
6.  Nuclei DAPI stained
7.  Nuclei only
8.  Median Bodies only
9.  Axoneme only
10. Median bodies and axoneme
11. Nuclei and axoneme
12. Nuclei and median bodies
13. Nuclei, median bodies and axoneme

narration: Select the characteristics from the list which indicate that the organisms are Giardia cysts. Select the “Check Answers” button when finished. FITC, DIC and DAPI pictures of Giardia



Characterization of Cryptosporidium: FITC

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Accurate and precise detection is imperative for Cryptosporidium oocyst enumeration.

Cryptosporidium under FITC exhibiting brightly highlighted rim

Record observations on the Cryptosporidium Examination Results Form.

Note IconTricks 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

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Cryptosporidium Results Examination Form highlighting shape column 2 pictures, one of round Cryptosporidum and second of oval



Tips and Tricks

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Characterization of Cryptosporidium: DAPI

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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.

Note IconTricks 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.

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Cryptosporidium Examination Results Form

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Cryptosporidium Results Examination  Form highlighting all 3 DAPI columns 2 pictures, one of round Cryptosporidium and second of oval



Tips and Tricks

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Characterization of Cryptosporidium: DIC

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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.

Note IconTricks 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.

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Cryptosporidium Examination Results Form

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DIC observations are recorded in the 3 DIC columns on the Cryptosporidium Examination Results Form.


Cryptosporidum Examination Results Form with empty and amorphous recorded as presence/absence and sporozoites recorded as a number 2 pictures, one of round Cryptosporidium and second of oval



Tips and Tricks

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Giardia and Cryptosporidium Measurement under DIC

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All size measurements must be determined using 1000x magnification under DIC and reported to the nearest 0.5 micrometers (µm).

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 with micrometers



Giardia and Cryptosporidium Examination Results Forms

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Giardia Examination Results Form with the size column highlighted Cryptosporidium Examination Results Form with the size column highlighted




Knowledge Review: Cryptosporidium

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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.

  1. One sporozoite
  2. Correct shape
  3. Amorphous internal structure
  4. DAPI negative
  5. Nuclei DAPI stained
  6. Brilliant apple-green fluorescence
  7. Three sporozoites
A. B. C.


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. Cryptosporidium



Review of FITC, DAPI and DIC

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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.

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Variation of Giardia: FITC

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typical fluorescence pattern







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. variations of Giardia



Variation of Giardia:DAPI

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Variations in Giardia

*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

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typical pattern

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.

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Knowledge Review: Giardia

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Mark the characteristic(s) which is(are) depicted in the DIC and/or DAPI photographs of the same organism:

Organisms for Knowledge Review

1. Nuclei
2. Median Bodies
3. Axonemes
4. DAPI stained nuclei
5. Cytoplasm pulled away from wall
6. Amorphous internal structure
7. DAPI negative
8. DAPI intense internal blue staining

narration: Select the characteristics which are observed in both photographs of Giardia; then select the "Check Answers" button.



Variation of Cryptosporidium:FITC

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typical pattern






variations of Cryptosporidium
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

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variations of Cryptosporidium: DAPI

*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

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Typical pattern o
f 4 distinct sporozoites

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

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View the video sample for sporozoites in the oocysts. Sporozoites are the most distinct in the three oocysts marked by arrows.


ideal pattern



Knowledge Review: Cryptosporidium

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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.)

Cryptosporidium

1. One sporozoite
2. Correct shape – round to oval
3. Amorphous internal structure
4. "Pac Man" look
5. Nuclei DAPI stained
6. Brilliant apple-green fluorescence
7. Two sporozoites
8. Three sporozoites
9. Wrinkled
10. Folded


narration: The photographs depict three possible Cryptosporidium oocysts. Choose the diagnostic features observed that indicate the organisms are oocysts.



Characterization of Interfering Organisms

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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:

Organisms similar to Cryptosporidium and Giardia
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

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The following are features of organisms observed with FITC fluorescence which help the analyst to differentiate the organism from Giardia:

Giardia fluorescent pattern
Giardia - like FITC
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

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The following are features of organisms observed with DAPI fluorescence that indicate the organism is not Giardia:
Giardia - DAPI positiv
e

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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.

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Interfering Organisms: Giardia-like DIC

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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
  • Giardia internal morphology
  • 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.

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Knowledge Review: Giardia

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Is it Giardia? Check the characteristics observed in the photos which support why you think the organism(s) is(are) or is(are) not Giardia. Once you have decided, check the Yes or No box below. (Photos depict the same organism.)
1. Nuclei
2. Median Bodies
3. Axonemes
4. Atypical internal structure
5. One large nucleus observable on DIC
6. Multiple cells
7. Cytoplasm pulled away from wall
8. Incorrect shape
9. Correct shape
10. Brightly highlighted cell wall
11. Fluorescence intensity similar between the rim and interior
12. DAPI stained nuclei
13. Intense blue internal staining with DAPI
14. DAPI negative
15. Cell wall exhibits correct structure on DIC
Organisms for Knowledge Review

   Yes, it is Giardia.
   No, it is not Giardia.

   

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

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typical pattern

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. interfering organisms



Cryptosporidium-like FITC

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Compare the typical FITC fluorescence pattern to the FITC fluorescence of interfering organisms.


typical pattern interfering organisms



Interfering Organisms: Cryptosporidium-like DAPI

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typical pattern

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

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Compare the typical DAPI positive pattern to the DAPI pattern of interfering organisms.


typical and interfering DAPI



Interfering Organisms: Cryptosporidium-like DIC

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  • 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


ideal pattern
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

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Compare the ideal pattern of the wall and sporozoites with the patterns observed in interfering organisms.


ideal pattern



Knowledge Review: Cryptosporidium

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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.)
1. One sporozoite
2. Amorphous internal structure
3. Atypical internal morphology on DIC
4. Atypical shape
5. Correct shape
6. Atypical cell wall on DIC
7. Typical cell wall on DIC
8. Atypical fluorescent pattern
9. Brilliant apple-green fluorescence
10. Four or fewer DAPI stained nuclei
11. More than four DAPI stained nuclei
12. Atypical DAPI staining
Organism for Knowledge Review




Yes, it is Cryptosporidium.
No, it is not Cryptosporidium.


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

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Is it Giardia? Check the characteristics observed in the photos which support why you think the organism is or is not Giardia. Once you have decided, check the Yes or No box below. (Photographs depict the same organism at maximum contrast and resolution.)
1. Nuclei
2. Median Bodies
3. Axonemes
4. Atypical internal structure
5. Amorphous internal morphology
6. Multiple cells
7. Cytoplasm pulled away from wall
8. Cell wall exhibits correct structure on DIC
9. Atypical shape
10. Typical shape
11. Brightly highlighted cell wall
12. Fluorescence intensity similar between the rim and interior
13. Atypical fluorescent pattern
14. DAPI stained nuclei
15. Intense blue internal staining with DAPI
16. DAPI negative
17. Atypical DAPI staining
Organism for Knowledge Review

Yes, it is Giardia.
No, it is not Giardia.


narration: A possible Giardia cyst is shown in these three photos. Select the observable characteristics which support whether it is or is not a cyst.



Enumerating a Field/Environmental Slide: Examination Results Form

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The microscope examination form includes a summary of the number of organisms found in a given sample along with all of the descriptions. Note that object 4 was crossed out with a single line, initialed and dated because its characteristics were atypical.


narration: The results of enumerating cysts and oocysts in a field slide are recorded on the examination results form. Note the sample information is enough to track the client and the sample. The bottom of the table contains a summary of the various DAPI and DIC categories. The Total FA number represents all the organisms which fluoresce a brilliant apple-green, are the appropriate size and shape, and have no atypical characteristics when viewed with FITC, DAPI or DIC. Be sure to cross out any atypical objects. Examination Results Form



Knowledge Review

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Which structure of Giardia will not be observed in any other organism?


A. Wall
B. Axonemes
C. Nuclei
D. Median bodies

narration: Choose the structure which is not observed in any other organism.



Knowledge Review

crypto0410 | page 36 of 40

Is it Cryptosporidium?

Check the characteristics observed in the photos which support why you think the organism is or is not Cryptosporidium. Once you have decided, check the Yes or No box below. (FITC and DIC photographs represent the same organism)

1. Atypical internal morphology on DIC
2. One sporozoite
3. Three sporozoites
4. Amorphous internal structure
5. Cell wall observable on DIC
6. Atypical fluorescent pattern
7. Fluorescence is crisp and sharp
8. Brilliant apple-green fluorescence
9. Atypical shape
10. Correct shape

Yes, it is Cryptosporidium.
No, it is not Cryptosporidium.

organisms for Knowledge Review

narration: Use the photographs and list of characteristics to decide if the organism shown is Cryptosporidium or not. Select the “Check Answers” button when finished.



Knowledge Review

crypto0440 | page 37 of 40

Is it Cryptosporidium?

Check the characteristics observed in the photos which support why you think the organism is or is not Cryptosporidium. Once you have decided, check the Yes or No box below. (assume photographs represent the same organism and microscope is adjusted for best contrast and resolution)

1. One sporozoite
2. Three sporozoites
3. No apparent cell wall
4. Atypical internal morphology on DIC
5. Atypical shape
6. Correct shape
7. Atypical fluorescence
8. Brilliant apple-green fluorescence
9. Fluorescence is not crisp and sharp
10. Fluorescence is crisp and sharp
11. More than 4 DAPI stained nuclei
12. Atypical DAPI staining
13. Cell wall on DAPI atypical color
organisms for Knowledge Review
Yes, it is Cryptosporidium.
No, it is not Cryptosporidium.


narration: Again, decide if the organism shown is an oocyst or not. Select the appropriate characteristics to support the choice and then check the answer.



Summary (1 of 2)

crypto0450 | page 38 of 40

After completing this module, the analyst should be able to describe a positive control Giardia cyst and Cryptosporidium oocyst based on FITC and DAPI fluorescence and DIC.

The classic Giardia cyst displays:

The classic Cryptosporidium oocyst displays:


narration: Congratulations! You have just completed the module on Giardia and Cryptosporidium. You should now be able to describe a positive control Giardia cyst and Cryptosporidium oocyst based on FITC and DAPI fluorescence and DIC. Remember all the characteristics are not observable all the time.



Summary (2 of 2)

crypto0460 | page 39 of 40

The determination of cross-reacting organisms is one of the most important skills of an analyst. It takes many years of experience examining many different water matrices to be proficient. It is always helpful to discuss the characteristics of the organisms with another analyst (e.g. looking through the same microscope or sharing photographs or video at conferences). If another analyst is not available, examining various photographs helps.

Features indicating a cross-reacting organism:

Some field samples are very challenging and will take time to complete. The goal of every analyst is:


narration: The following continuation of training is recommended: review this module a number of times, compare the photographs to organisms observed at the microscope, spend time at the microscope at least a few days a week to learn the characteristics, and photograph organisms to document observations of both positive control and interfering organism characteristics.



Acknowledgements/Contacts

crypto0470 | page 40 of 40

EPA wishes to thank the following utilities and laboratories for donating photographs for use in the module (alphabetical order):
ACH Diagnostic & Consulting Service, Inc.
BCity of San Diego Water Quality Laboratory
CCity of Tampa, J.T. Gordy
DEPA, NHSRC, Office of Research and Development, H.D.A. Lindquist
EEPA, NRML, Office of Research and Development, F.P. Williams, Jr.
FEPA, Office of Ground Water and Drinking Water, TSC
GEPCOR, P. Poon
HLas Vegas Valley Water System, C.J. Meyer
ILouisville Water Company, R. Tucker
JMohawk Valley Water Authority, C. Schreppel
KNew York City DEP Pathogen Laboratory
LOrange County Utility Laboratory (FL), D. Vaughn
MSan Francisco Public Utilities Commission - Water Quality Parasite Laboratory
NSanta Clara Valley Water District, Laboratory Services
OTexas Agricultural Experiment Station - El Paso, Texas A&M University, G.D. Di Giovanni
PUniversity of Iowa Hygienic Laboratory
Qhttp://idsc.nih.go.jp/index.html
For questions, contact EPA, Office of Ground Water and Drinking Water, Technical Support Center at
miller.carrie@epa.gov.
This completes the module.
narration: Thank you to all the utilities and laboratories listed for the photographs used in this module. The listed laboratories have granted permission to EPA to use the photographs. Please request permission and credit the appropriate photographer if any of these photographs are used for any publicly distributed media.